RESUMEN
The kidney is an essential organ that ensures bodily fluid homeostasis and removes soluble waste products from the organism. Nephrons, the functional units of the kidney, comprise a blood filter, the glomerulus or glomus, and an epithelial tubule that processes the filtrate from the blood or coelom and selectively reabsorbs solutes, such as sugars, proteins, ions, and water, leaving waste products to be eliminated in the urine. Genes coding for transporters are segmentally expressed, enabling the nephron to sequentially process the filtrate. The Xenopus embryonic kidney, the pronephros, which consists of a single large nephron, has served as a valuable model to identify genes involved in nephron formation and patterning. Therefore, the developmental patterning program that generates these segments is of great interest. Prior work has defined the gene expression profiles of Xenopus nephron segments via in situ hybridization strategies, but a comprehensive understanding of the cellular makeup of the pronephric kidney remains incomplete. Here, we carried out single-cell mRNA sequencing of the functional Xenopus pronephric nephron and evaluated its cellular composition through comparative analyses with previous Xenopus studies and single-cell mRNA sequencing of the adult mouse kidney. This study reconstructs the cellular makeup of the pronephric kidney and identifies conserved cells, segments, and associated gene expression profiles. Thus, our data highlight significant conservation in podocytes, proximal and distal tubule cells, and divergence in cellular composition underlying the capacity of each nephron to remove wastes in the form of urine, while emphasizing the Xenopus pronephros as a model for physiology and disease.
Asunto(s)
Riñón , Nefronas , Animales , Ratones , Regulación del Desarrollo de la Expresión Génica , Riñón/embriología , Glomérulos Renales/embriología , Nefronas/embriología , ARN Mensajero/genética , Xenopus laevis/embriologíaRESUMEN
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 âkb) 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.
Asunto(s)
Proteínas ADAMTS/genética , Proteínas ADAMTS/metabolismo , Uréter/embriología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Femenino , Riñón/metabolismo , Riñón/patología , Masculino , Mamíferos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Morfogénesis/genética , Nefronas/metabolismo , Organogénesis/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Uréter/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Genetic factors underlying the human limb abnormality congenital talipes equinovarus ('clubfoot') remain incompletely understood. The spontaneous autosomal recessive mouse 'peroneal muscular atrophy' mutant (PMA) is a faithful morphological model of human clubfoot. In PMA mice, the dorsal (peroneal) branches of the sciatic nerves are absent. In this study, the primary developmental defect was identified as a reduced growth of sciatic nerve lateral motor column (LMC) neurons leading to failure to project to dorsal (peroneal) lower limb muscle blocks. The pma mutation was mapped and a candidate gene encoding LIM-domain kinase 1 (Limk1) identified, which is upregulated in mutant lateral LMC motor neurons. Genetic and molecular analyses showed that the mutation acts in the EphA4-Limk1-Cfl1/cofilin-actin pathway to modulate growth cone extension/collapse. In the chicken, both experimental upregulation of Limk1 by electroporation and pharmacological inhibition of actin turnover led to defects in hindlimb spinal motor neuron growth and pathfinding, and mimicked the clubfoot phenotype. The data support a neuromuscular aetiology for clubfoot and provide a mechanistic framework to understand clubfoot in humans.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/embriología , Pie Equinovaro/embriología , Pie Equinovaro/genética , Quinasas Lim/genética , Mutación , Animales , Axones , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/patología , Embrión de Pollo , Mapeo Cromosómico , Pie Equinovaro/patología , Modelos Animales de Enfermedad , Femenino , Miembro Posterior/anomalías , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Neuronas Motoras/patología , Músculo Esquelético/anomalías , Músculo Esquelético/inervación , Nervio Peroneo/anomalías , Fenotipo , Embarazo , Receptor EphA4/deficiencia , Receptor EphA4/genética , Nervio Ciático/anomalías , Regulación hacia ArribaRESUMEN
The nephron is the functional unit of the kidney, but the mechanism of nephron formation during human development is unclear. We conducted a detailed analysis of nephron development in humans and mice by immunolabeling, and we compared human and mouse nephron patterning to describe conserved and divergent features. We created protein localization maps that highlight the emerging patterns along the proximal-distal axis of the developing nephron and benchmark expectations for localization of functionally important transcription factors, which revealed unanticipated cellular diversity. Moreover, we identified a novel nephron subdomain marked by Wnt4 expression that we fate-mapped to the proximal mature nephron. Significant conservation was observed between human and mouse patterning. We also determined the time at which markers for mature nephron cell types first emerge-critical data for the renal organoid field. These findings have conceptual implications for the evolutionary processes driving the diversity of mammalian organ systems. Furthermore, these findings provide practical insights beyond those gained with mouse and rat models that will guide in vitro efforts to harness the developmental programs necessary to build human kidney structures.
Asunto(s)
Diferenciación Celular , Nefronas/embriología , Nefronas/metabolismo , Células Madre/fisiología , Animales , Proteínas Reguladoras de la Apoptosis , Linaje de la Célula , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Inmunohistoquímica , Factor de Unión 1 al Potenciador Linfoide/genética , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células Madre/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína Wnt4/metabolismoRESUMEN
Cellular interactions among nephron, interstitial, and collecting duct progenitors drive mammalian kidney development. In mice, Six2+ nephron progenitor cells (NPCs) and Foxd1+ interstitial progenitor cells (IPCs) form largely distinct lineage compartments at the onset of metanephric kidney development. Here, we used the method for analyzing RNA following intracellular sorting (MARIS) approach, single-cell transcriptional profiling, in situ hybridization, and immunolabeling to characterize the presumptive NPC and IPC compartments of the developing human kidney. As in mice, each progenitor population adopts a stereotypical arrangement in the human nephron-forming niche: NPCs capped outgrowing ureteric branch tips, whereas IPCs were sandwiched between the NPCs and the renal capsule. Unlike mouse NPCs, human NPCs displayed a transcriptional profile that overlapped substantially with the IPC transcriptional profile, and key IPC determinants, including FOXD1, were readily detected within SIX2+ NPCs. Comparative gene expression profiling in human and mouse Six2/SIX2+ NPCs showed broad agreement between the species but also identified species-biased expression of some genes. Notably, some human NPC-enriched genes, including DAPL1 and COL9A2, are linked to human renal disease. We further explored the cellular diversity of mesenchymal cell types in the human nephrogenic niche through single-cell transcriptional profiling. Data analysis stratified NPCs into two main subpopulations and identified a third group of differentiating cells. These findings were confirmed by section in situ hybridization with novel human NPC markers predicted through the single-cell studies. This study provides a benchmark for the mesenchymal progenitors in the human nephrogenic niche and highlights species-variability in kidney developmental programs.
Asunto(s)
Corteza Renal/embriología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Nefronas/embriología , Animales , Proteínas Reguladoras de la Apoptosis , Diferenciación Celular , Linaje de la Célula , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Ratones , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/genética , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/metabolismo , Nefronas/anatomía & histología , Nefronas/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Human kidney function is underpinned by approximately 1,000,000 nephrons, although the number varies substantially, and low nephron number is linked to disease. Human kidney development initiates around 4 weeks of gestation and ends around 34-37 weeks of gestation. Over this period, a reiterative inductive process establishes the nephron complement. Studies have provided insightful anatomic descriptions of human kidney development, but the limited histologic views are not readily accessible to a broad audience. In this first paper in a series providing comprehensive insight into human kidney formation, we examined human kidney development in 135 anonymously donated human kidney specimens. We documented kidney development at a macroscopic and cellular level through histologic analysis, RNA in situ hybridization, immunofluorescence studies, and transcriptional profiling, contrasting human development (4-23 weeks) with mouse development at selected stages (embryonic day 15.5 and postnatal day 2). The high-resolution histologic interactive atlas of human kidney organogenesis generated can be viewed at the GUDMAP database (www.gudmap.org) together with three-dimensional reconstructions of key components of the data herein. At the anatomic level, human and mouse kidney development differ in timing, scale, and global features such as lobe formation and progenitor niche organization. The data also highlight differences in molecular and cellular features, including the expression and cellular distribution of anchor gene markers used to identify key cell types in mouse kidney studies. These data will facilitate and inform in vitro efforts to generate human kidney structures and comparative functional analyses across mammalian species.
Asunto(s)
Riñón/embriología , Riñón/metabolismo , Organogénesis , Uréter/embriología , Animales , Diferenciación Celular , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Edad Gestacional , Técnicas Histológicas , Humanos , Hibridación in Situ , Riñón/anatomía & histología , Ratones , Nefronas/embriología , Nefronas/metabolismo , ARN/análisis , Uréter/metabolismoRESUMEN
This report presents a novel mechanism for remodelling a branched epithelial tree. The mouse renal collecting duct develops by growth and repeated branching of an initially unbranched ureteric bud: this mechanism initially produces an almost fractal form with young branches connected to the centre of the kidney via a sequence of nodes (branch points) distributed widely throughout the developing organ. The collecting ducts of a mature kidney have a different form: from the nephrons in the renal cortex, long, straight lengths of collecting duct run almost parallel to one another through the renal medulla, and open together to the renal pelvis. Here we present time-lapse studies of E11.5 kidneys growing in culture: after about 5 days, the collecting duct trees show evidence of 'node retraction', in which the node of a 'Y'-shaped branch moves downwards, shortening the stalk of the 'Y', lengthening its arms and narrowing their divergence angle so that the 'Y' becomes a 'V'. Computer simulation suggests that node retraction can transform a spread tree, like that of an early kidney, into one with long, almost-parallel medullary rays similar to those seen in a mature real kidney.
Asunto(s)
Túbulos Renales Colectores/embriología , Modelos Biológicos , Morfogénesis/fisiología , Animales , Técnicas de Cultivo de Célula , Células Cultivadas , Simulación por Computador , Ratones , Ratones Transgénicos , Imagen de Lapso de TiempoAsunto(s)
Enfermedades Renales , Transcriptoma , Animales , Catalogación , Humanos , Ratones , Análisis de Secuencia de ARNRESUMEN
The mammalian kidney maintains fluid homeostasis through diverse epithelial cell types generated from nephron and ureteric progenitor cells. To extend a developmental understanding of the kidney's epithelial networks, we compared chromatin organization (single-nuclear assay for transposase-accessible chromatin sequencing [ATAC-seq]; 112,864 nuclei) and gene expression (single-cell/nuclear RNA sequencing [RNA-seq]; 109,477 cells/nuclei) in the developing human (10.6-17.6 weeks; n = 10) and mouse (post-natal day [P]0; n = 10) kidney, supplementing analysis with published mouse datasets from earlier stages. Single-cell/nuclear datasets were analyzed at a species level, and then nephron and ureteric cellular lineages were extracted and integrated into a common, cross-species, multimodal dataset. Comparative computational analyses identified conserved and divergent features of chromatin organization and linked gene activity, identifying species-specific and cell-type-specific regulatory programs. In situ validation of human-enriched gene activity points to human-specific signaling interactions in kidney development. Further, human-specific enhancer regions were linked to kidney diseases through genome-wide association studies (GWASs), highlighting the potential for clinical insight from developmental modeling.
RESUMEN
The kidney maintains body fluid homeostasis by reabsorbing essential compounds and excreting waste. Proximal tubule cells, crucial for renal reabsorption of a range of sugars, ions, and amino acids, are highly susceptible to damage, leading to pathologies necessitating dialysis and kidney transplants. While human pluripotent stem cell-derived kidney organoids are used for modeling renal development, disease, and injury, the formation of proximal nephron cells in these 3D structures is incomplete. Here, we describe how to drive the development of proximal tubule precursors in kidney organoids by following a blueprint of in vivo human nephrogenesis. Transient manipulation of the PI3K signaling pathway activates Notch signaling in the early nephron and drives nephrons toward a proximal precursor state. These "proximal-biased" (PB) organoid nephrons proceed to generate proximal nephron precursor cells. Single-cell transcriptional analyses across the organoid nephron differentiation, comparing control and PB types, confirm the requirement of transient Notch signaling for proximal development. Indicative of functional maturity, PB organoids demonstrate dextran and albumin uptake, akin to in vivo proximal tubules. Moreover, PB organoids are highly sensitive to nephrotoxic agents, display an injury response, and drive expression of HAVCR1 / KIM1 , an early proximal-specific marker of kidney injury. Injured PB organoids show evidence of collapsed tubules, DNA damage, and upregulate the injury-response marker SOX9 . The PB organoid model therefore has functional relevance and potential for modeling mechanisms underpinning nephron injury. These advances improve the use of iPSC-derived kidney organoids as tools to understand developmental nephrology, model disease, test novel therapeutics, and for understanding human renal physiology.
RESUMEN
Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here, manipulation of p38 and YAP activity allowed for long-term clonal expansion of primary mouse and human NPCs and induced NPCs (iNPCs) from human pluripotent stem cells (hPSCs). Molecular analyses demonstrated that cultured iNPCs closely resemble primary human NPCs. iNPCs generated nephron organoids with minimal off-target cell types and enhanced maturation of podocytes relative to published human kidney organoid protocols. Surprisingly, the NPC culture medium uncovered plasticity in human podocyte programs, enabling podocyte reprogramming to an NPC-like state. Scalability and ease of genome editing facilitated genome-wide CRISPR screening in NPC culture, uncovering genes associated with kidney development and disease. Further, NPC-directed modeling of autosomal-dominant polycystic kidney disease (ADPKD) identified a small-molecule inhibitor of cystogenesis. These findings highlight a broad application for the reported iNPC platform in the study of kidney development, disease, plasticity, and regeneration.
Asunto(s)
Nefronas , Organoides , Animales , Organoides/citología , Organoides/metabolismo , Humanos , Nefronas/citología , Ratones , Diferenciación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Podocitos/metabolismo , Podocitos/citología , Riñón/patología , Riñón Poliquístico Autosómico Dominante/patología , Riñón Poliquístico Autosómico Dominante/metabolismo , Riñón Poliquístico Autosómico Dominante/genética , Modelos Biológicos , Edición GénicaRESUMEN
Wnt regulated transcriptional programs are associated with both the maintenance of mammalian nephron progenitor cells (NPC) and their induction, initiating the process of nephrogenesis. How opposing transcriptional roles are regulated remain unclear. Using an in vitro model replicating in vivo events, we examined the requirement for canonical Wnt transcriptional complexes in NPC regulation. In canonical transcription, Lef/Tcf DNA binding proteins associate the transcriptional co-activator ß-catenin. Wnt signaling is readily substituted by CHIR99021, a small molecule antagonist of glycogen synthase kinase-3ß (GSK3ß). GSK3ß inhibition blocks Gskß-dependent turnover of ß-catenin, enabling formation of Lef/Tcf/ß-catenin transcriptional complexes, and enhancer-mediated transcriptional activation. Removal of ß-catenin activity from NPCs under cell expansion conditions (low CHIR) demonstrated a non-transcriptional role for ß-catenin in the CHIR-dependent proliferation of NPCs. In contrast, CHIR-mediated induction of nephrogenesis, on switching from low to high CHIR, was dependent on Lef/Tcf and ß-catenin transcriptional activity. These studies point to a non-transcriptional mechanism for ß-catenin in regulation of NPCs, and potentially other stem progenitor cell types. Further, analysis of the ß-catenin-directed transcriptional response provides new insight into induction of nephrogenesis. Summary Statement: The study provides a mechanistic understanding of Wnt/ ß-catenin activity in self-renewal and differentiation of mammalian nephron progenitors.
RESUMEN
Mammalian kidneys maintain fluid homeostasis through the cellular activity of nephrons and the conjoined collecting system. Each epithelial network originates from distinct progenitor cell populations that reciprocally interact during development. To extend our understanding of human and mouse kidney development, we profiled chromatin organization (ATAC-seq) and gene expression (RNA-seq) in developing human and mouse kidneys. Data were analyzed at a species level and then integrated into a common, cross-species multimodal data set. Comparative analysis of cell types and developmental trajectories identified conserved and divergent features of chromatin organization and linked gene activity, revealing species- and cell-type specific regulatory programs. Identification of human-specific enhancer regions linked through GWAS studies to kidney disease highlights the potential of developmental modeling to provide clinical insight.
RESUMEN
Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here we report manipulation of p38 and YAP activity creates a synthetic niche that allows the long-term clonal expansion of primary mouse and human NPCs, and induced NPCs (iNPCs) from human pluripotent stem cells. Cultured iNPCs resemble closely primary human NPCs, generating nephron organoids with abundant distal convoluted tubule cells, which are not observed in published kidney organoids. The synthetic niche reprograms differentiated nephron cells into NPC state, recapitulating the plasticity of developing nephron in vivo. Scalability and ease of genome-editing in the cultured NPCs allow for genome-wide CRISPR screening, identifying novel genes associated with kidney development and disease. A rapid, efficient, and scalable organoid model for polycystic kidney disease was derived directly from genome-edited NPCs, and validated in drug screen. These technological platforms have broad applications to kidney development, disease, plasticity, and regeneration.
RESUMEN
Human developmental studies and regenerative therapies need in vitro systems that generate cell types recapitulating mature cell physiologies. In a recent issue of Nature Biotechnology, Shi et al. (2022) show how pluripotent stem cells can differentiate into ureteric bud organoids that mature into functional kidney collecting duct cell types.
Asunto(s)
Riñón , Células Madre Pluripotentes , Diferenciación Celular/fisiología , Humanos , Riñón/citología , Riñón/metabolismo , OrganoidesRESUMEN
Human pluripotent stem-cell-derived organoids are models for human development and disease. We report a modified human kidney organoid system that generates thousands of similar organoids, each consisting of 1-2 nephron-like structures. Single-cell transcriptomic profiling and immunofluorescence validation highlighted patterned nephron-like structures utilizing similar pathways, with distinct morphogenesis, to human nephrogenesis. To examine this platform for therapeutic screening, the polycystic kidney disease genes PKD1 and PKD2 were inactivated by gene editing. PKD1 and PKD2 mutant models exhibited efficient and reproducible cyst formation. Cystic outgrowths could be propagated for months to centimeter-sized cysts. To shed new light on cystogenesis, 247 protein kinase inhibitors (PKIs) were screened in a live imaging assay identifying compounds blocking cyst formation but not overall organoid growth. Scaling and further development of the organoid platform will enable a broader capability for kidney disease modeling and high-throughput drug screens.
Asunto(s)
Quistes , Riñón Poliquístico Autosómico Dominante , Quistes/metabolismo , Descubrimiento de Drogas , Humanos , Riñón/metabolismo , Organoides/metabolismo , Riñón Poliquístico Autosómico Dominante/tratamiento farmacológico , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/metabolismo , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismoRESUMEN
Congenital abnormalities of the kidney and urinary tract are among the most common birth defects, affecting 3% of newborns. The human kidney forms around a million nephrons from a pool of nephron progenitors over a 30-week period of development. To establish a framework for human nephrogenesis, we spatially resolved a stereotypical process by which equipotent nephron progenitors generate a nephron anlage, then applied data-driven approaches to construct three-dimensional protein maps on anatomical models of the nephrogenic program. Single-cell RNA sequencing identified progenitor states, which were spatially mapped to the nephron anatomy, enabling the generation of functional gene networks predicting interactions within and between nephron cell types. Network mining identified known developmental disease genes and predicted targets of interest. The spatially resolved nephrogenic program made available through the Human Nephrogenesis Atlas (https://sckidney.flatironinstitute.org/) will facilitate an understanding of kidney development and disease and enhance efforts to generate new kidney structures.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Nefronas/metabolismo , Transcriptoma , Animales , Humanos , Ratones , Nefronas/citología , Nefronas/embriología , Proteoma/genética , Proteoma/metabolismo , RNA-Seq , Análisis de la Célula IndividualRESUMEN
The overall pattern of the developing kidney is set in large part by the developing ureteric bud/collecting duct system, and dysgenesis of this system accounts for a variety of clinically significant renal diseases. Understanding how the behavior of cells in the developing ureteric bud/collecting duct is controlled is therefore important to understanding the normal and abnormal kidney. Dact proteins have recently been identified as cytoplasmic regulators of intracellular signaling. Dact1 inhibits Wnt signaling, and Dact2 inhibits transforming growth factor (TGF)-ß signaling. Here, we report that Dact2 is expressed in developing and adult mouse kidneys, specifically in the ureteric bud/collecting duct epithelium, a structure whose morphogenesis is controlled partially by TGF-ß. When small interfering RNA is used to knock down Dact2 expression in collecting duct cells, they show some constitutive phospho-Smad2, undetectable in controls, and elevated phospho-Smad2 in response to TGF-ß. They also show defective migration and, in a monolayer wound-healing assay, they fail to assemble a leading edge "cable" of actomyosin and advance instead as a disorganized mass of lamellipodium-bearing cells. This effect is seriously exacerbated by exogenous TGF-ß, although control cells tolerate it well. In three-dimensional culture, Dact2 knockdown cells form cysts and branching tubules, but the outlines of the cysts made by knockdown cells are ragged rather than smooth and the branching tubules are decorated with many fine spikes not seen in controls. These data suggest Dact2 plays a role in regulating morphogenesis by renal collecting duct cells, probably by protecting cells from overly strong TGF-ß pathway activation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Túbulos Renales Colectores/embriología , Túbulos Renales Colectores/metabolismo , Riñón/metabolismo , Morfogénesis/fisiología , Uréter/embriología , Uréter/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular , Movimiento Celular , Riñón/embriología , Túbulos Renales Colectores/citología , Ratones , Ratones Noqueados , ARN Mensajero/metabolismo , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
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.
Asunto(s)
Linaje de la Célula , Riñón/citología , Caracteres Sexuales , Análisis de la Célula Individual , Animales , Células Epiteliales/citología , Femenino , Riñón/anatomía & histología , Masculino , Ratones , Nefronas/citología , Factores de TiempoRESUMEN
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.