ABSTRACT
Isthmin-1 (Ism1) was first described to be syn-expressed with Fgf8 in Xenopus. However, its biological role has not been elucidated until recent years. Despite of accumulated evidence that Ism1 participates in angiogenesis, tumor invasion, macrophage apoptosis, and glucose metabolism, the cognate receptors for Ism1 remain largely unknown. Ism1 deficiency in mice results in renal agenesis (RA) with a transient loss of Gdnf transcription and impaired mesenchyme condensation at E11.5. Ism1 binds to and activates Integrin α8ß1 to positively regulate Gdnf/Ret signaling, thus promoting mesenchyme condensation and ureteric epithelium branching morphogenesis. Here, we propose the hypothesis underlying the mechanism by which Ism1 regulates branching morphogenesis during early kidney development.
Subject(s)
Embryonic Structures , Glial Cell Line-Derived Neurotrophic Factor , Nephrons/embryology , Ureter , Mice , Animals , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Kidney/abnormalities , Kidney/metabolism , Kidney/pathology , Ureter/metabolism , MorphogenesisABSTRACT
Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below age 30 years. Many monogenic forms have been discovered due to comprehensive genetic testing like exome sequencing. However, disease-causing variants in known disease-associated genes only explain a proportion of cases. Here, we aim to unravel underlying molecular mechanisms of syndromic CAKUT in three unrelated multiplex families with presumed autosomal recessive inheritance. Exome sequencing in the index individuals revealed three different rare homozygous variants in FOXD2, encoding a transcription factor not previously implicated in CAKUT in humans: a frameshift in the Arabic and a missense variant each in the Turkish and the Israeli family with segregation patterns consistent with autosomal recessive inheritance. CRISPR/Cas9-derived Foxd2 knockout mice presented with a bilateral dilated kidney pelvis accompanied by atrophy of the kidney papilla and mandibular, ophthalmologic, and behavioral anomalies, recapitulating the human phenotype. In a complementary approach to study pathomechanisms of FOXD2-dysfunction-mediated developmental kidney defects, we generated CRISPR/Cas9-mediated knockout of Foxd2 in ureteric bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important for kidney/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a shift toward a stromal cell identity. Histology of Foxd2 knockout mouse kidneys confirmed increased fibrosis. Further, genome-wide association studies suggest that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Thus, our studies help in genetic diagnostics of monogenic CAKUT and in understanding of monogenic and multifactorial kidney diseases.
Subject(s)
Embryonic Structures , Forkhead Transcription Factors , Kidney Diseases , Kidney , Nephrons , Urinary Tract , Urogenital Abnormalities , Vesico-Ureteral Reflux , Adult , Animals , Humans , Mice , Genome-Wide Association Study , Kidney/abnormalities , Kidney/embryology , Kidney Diseases/genetics , Mice, Knockout , Nephrons/embryology , Transcription Factors/genetics , Urogenital Abnormalities/genetics , Vesico-Ureteral Reflux/genetics , Forkhead Transcription Factors/deficiency , Forkhead Transcription Factors/metabolismABSTRACT
Nephron endowment, defined during the fetal period, dictates renal and related cardiovascular health throughout life. We show here that, despite its negative effects on kidney growth, genetic increase of GDNF prolongs the nephrogenic program beyond its normal cessation. Multi-stage mechanistic analysis revealed that excess GDNF maintains nephron progenitors and nephrogenesis through increased expression of its secreted targets and augmented WNT signaling, leading to a two-part effect on nephron progenitor maintenance. Abnormally high GDNF in embryonic kidneys upregulates its known targets but also Wnt9b and Axin2, with concomitant deceleration of nephron progenitor proliferation. Decline of GDNF levels in postnatal kidneys normalizes the ureteric bud and creates a permissive environment for continuation of the nephrogenic program, as demonstrated by morphologically and molecularly normal postnatal nephron progenitor self-renewal and differentiation. These results establish that excess GDNF has a bi-phasic effect on nephron progenitors in mice, which can faithfully respond to GDNF dosage manipulation during the fetal and postnatal period. Our results suggest that sensing the signaling activity level is an important mechanism through which GDNF and other molecules contribute to nephron progenitor lifespan specification.
Subject(s)
Glial Cell Line-Derived Neurotrophic Factor/metabolism , Nephrons/embryology , Nephrons/growth & development , Organogenesis/genetics , Wnt Signaling Pathway/genetics , Animals , Axin Protein/metabolism , Cell Differentiation/genetics , Glial Cell Line-Derived Neurotrophic Factor/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Stem Cells/cytology , Wnt Proteins/metabolismABSTRACT
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.
Subject(s)
Kidney , Nephrons , Animals , Mice , Gene Expression Regulation, Developmental , Kidney/embryology , Kidney Glomerulus/embryology , Nephrons/embryology , RNA, Messenger/genetics , Xenopus laevis/embryologyABSTRACT
There is an increasing interest in using zebrafish (Danio rerio) larva as a vertebrate screening model to study drug disposition. As the pronephric kidney of zebrafish larvae shares high similarity with the anatomy of nephrons in higher vertebrates including humans, we explored in this study whether 3- to 4-day-old zebrafish larvae have a fully functional pronephron. Intravenous injection of fluorescent polyethylene glycol and dextran derivatives of different molecular weight revealed a cutoff of 4.4-7.6 nm in hydrodynamic diameter for passive glomerular filtration, which is in agreement with corresponding values in rodents and humans. Distal tubular reabsorption of a FITC-folate conjugate, covalently modified with PEG2000, via folate receptor 1 was shown. Transport experiments of fluorescent substrates were assessed in the presence and absence of specific inhibitors in the blood systems. Thereby, functional expression in the proximal tubule of organic anion transporter oat (slc22) multidrug resistance-associated protein mrp1 (abcc1), mrp2 (abcc2), mrp4 (abcc4), and zebrafish larva p-glycoprotein analog abcb4 was shown. In addition, nonrenal clearance of fluorescent substrates and plasma protein binding characteristics were assessed in vivo. The results of transporter experiments were confirmed by extrapolation to ex vivo experiments in killifish (Fundulus heteroclitus) proximal kidney tubules. We conclude that the zebrafish larva has a fully functional pronephron at 96 h postfertilization and is therefore an attractive translational vertebrate screening model to bridge the gap between cell culture-based test systems and pharmacokinetic experiments in higher vertebrates.NEW & NOTEWORTHY The study of renal function remains a challenge. In vitro cell-based assays are approved to study, e.g., ABC/SLC-mediated drug transport but do not cover other renal functions such as glomerular filtration. Here, in vivo studies combined with in vitro assays are needed, which are time consuming and expensive. In view of these limitations, our proof-of-concept study demonstrates that the zebrafish larva is a translational in vivo test model that allows for mechanistic investigations to study renal function.
Subject(s)
Membrane Transport Proteins/metabolism , Nephrons/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Animals , Animals, Genetically Modified , Embryonic Development , Fluorescent Dyes/metabolism , Gene Expression Regulation, Developmental , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Larva/growth & development , Larva/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Membrane Transport Proteins/genetics , Microscopy, Confocal , Multidrug Resistance-Associated Protein 2/genetics , Multidrug Resistance-Associated Protein 2/metabolism , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Nephrons/embryology , Organic Cation Transport Proteins/genetics , Organic Cation Transport Proteins/metabolism , Proof of Concept Study , Time Factors , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/genetics , Red Fluorescent ProteinABSTRACT
Recent advances in the generation of kidney organoids and the culture of primary nephron progenitors from mouse and human have been based on knowledge of the molecular basis of kidney development in mice. Although gene expression during kidney development has been intensely investigated, single cell profiling provides new opportunities to further subsect component cell types and the signalling networks at play. Here, we describe the generation and analysis of 6732 single cell transcriptomes from the fetal mouse kidney [embryonic day (E)18.5] and 7853 sorted nephron progenitor cells (E14.5). These datasets provide improved resolution of cell types and specific markers, including subdivision of the renal stroma and heterogeneity within the nephron progenitor population. Ligand-receptor interaction and pathway analysis reveals novel crosstalk between cellular compartments and associates new pathways with differentiation of nephron and ureteric epithelium cell types. We identify transcriptional congruence between the distal nephron and ureteric epithelium, showing that most markers previously used to identify ureteric epithelium are not specific. Together, this work improves our understanding of metanephric kidney development and provides a template to guide the regeneration of renal tissue.
Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Developmental , Kidney/embryology , Receptor Cross-Talk , Single-Cell Analysis/methods , Algorithms , Animals , Cell Differentiation , Cell Lineage , Epithelium/embryology , Kidney/cytology , Ligands , Mice , Mice, Inbred C57BL , Nephrons/embryology , Organogenesis , Signal Transduction , Stem Cells/cytology , Transcriptome , Ureter/embryologyABSTRACT
Renal functional units known as nephrons undergo patterning events during development that create a segmental array of cellular compartments with discrete physiological identities. Here, from a forward genetic screen using zebrafish, we report the discovery that transcription factor AP-2 alpha (tfap2a) coordinates a gene regulatory network that activates the terminal differentiation program of distal segments in the pronephros. We found that tfap2a acts downstream of Iroquois homeobox 3b (irx3b), a distal lineage transcription factor, to operate a circuit consisting of tfap2b, irx1a and genes encoding solute transporters that dictate the specialized metabolic functions of distal nephron segments. Interestingly, this regulatory node is distinct from other checkpoints of differentiation, such as polarity establishment and ciliogenesis. Thus, our studies reveal insights into the genetic control of differentiation, where tfap2a is essential for regulating a suite of segment transporter traits at the final tier of zebrafish pronephros ontogeny. These findings have relevance for understanding renal birth defects, as well as efforts to recapitulate nephrogenesis in vivo to facilitate drug discovery and regenerative therapies.
Subject(s)
Kidney/embryology , Nephrons/embryology , Organogenesis/genetics , Transcription Factor AP-2/physiology , Zebrafish Proteins/physiology , Animals , Animals, Genetically Modified , Body Patterning/genetics , Cell Differentiation/genetics , Embryo, Nonmammalian , Gene Expression Regulation, Developmental , Genes, Switch/physiology , Kidney/metabolism , Nephrons/metabolism , Pronephros/embryology , Pronephros/growth & development , Pronephros/metabolism , Transcription Factor AP-2/genetics , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/geneticsABSTRACT
BACKGROUND: Most nephrons are added in late gestation. Truncated extrauterine nephrogenesis in premature infants results in fewer nephrons and significantly increased risk for CKD in adulthood. To overcome the ethical and technical difficulties associated with studies of late-gestation human fetal kidney development, third-trimester rhesus macaques served as a model to understand lateral branch nephrogenesis (LBN) at the molecular level. METHODS: Immunostaining and 3D rendering assessed morphology. Single-cell (sc) and single-nucleus (sn) RNA-Seq were performed on four cortically enriched fetal rhesus kidneys of 129-131 days gestational age (GA). An integrative bioinformatics strategy was applied across single-cell modalities, species, and time. RNAScope validation studies were performed on human archival tissue. RESULTS: Third-trimester rhesus kidney undergoes human-like LBN. scRNA-Seq of 23,608 cells revealed 37 transcriptionally distinct cell populations, including naïve nephron progenitor cells (NPCs), with the prior noted marker genes CITED1, MEOX1, and EYA1 (c25). These same populations and markers were reflected in snRNA-Seq of 5972 nuclei. Late-gestation rhesus NPC markers resembled late-gestation murine NPC, whereas early second-trimester human NPC markers aligned to midgestation murine NPCs. New, age-specific rhesus NPCs (SHISA8) and ureteric buds (POU3F4 and TWIST) predicted markers were verified in late-gestation human archival samples. CONCLUSIONS: Rhesus macaque is the first model of bona fide LBN, enabling molecular studies of late gestation, human-like nephrogenesis. These molecular findings support the hypothesis that aging nephron progenitors have a distinct molecular signature and align to their earlier human counterparts, with unique markers highlighting LBN-specific progenitor maturation.
Subject(s)
Models, Animal , Nephrons/embryology , Organogenesis/physiology , Animals , Fetus/anatomy & histology , Fetus/embryology , Fetus/metabolism , Gestational Age , Humans , Macaca mulatta , Stem Cells/physiologyABSTRACT
BACKGROUND: Low nephron number at birth is associated with a high risk of CKD in adulthood because nephrogenesis is completed in utero. Poor intrauterine environment impairs nephron endowment via an undefined molecular mechanism. A calorie-restricted diet (CRD) mouse model examined the effect of malnutrition during pregnancy on nephron progenitor cells (NPCs). METHODS: Daily caloric intake was reduced by 30% during pregnancy. mRNA expression, the cell cycle, and metabolic activity were evaluated in sorted Six2 NPCs. The results were validated using transgenic mice, oral nutrient supplementation, and organ cultures. RESULTS: Maternal CRD is associated with low nephron number in offspring, compromising kidney function at an older age. RNA-seq identified cell cycle regulators and the mTORC1 pathway, among other pathways, that maternal malnutrition in NPCs modifies. Metabolomics analysis of NPCs singled out the methionine pathway as crucial for NPC proliferation and maintenance. Methionine deprivation reduced NPC proliferation and lowered NPC number per tip in embryonic kidney cultures, with rescue from methionine metabolite supplementation. Importantly, in vivo, the negative effect of caloric restriction on nephrogenesis was prevented by adding methionine to the otherwise restricted diet during pregnancy or by removing one Tsc1 allele in NPCs. CONCLUSIONS: These findings show that mTORC1 signaling and methionine metabolism are central to the cellular and metabolic effects of malnutrition during pregnancy on NPCs, contributing to nephrogenesis and later, to kidney health in adulthood.
Subject(s)
Malnutrition/physiopathology , Mechanistic Target of Rapamycin Complex 1/metabolism , Methionine/metabolism , Nephrons/embryology , Stem Cells/metabolism , Animals , Caloric Restriction , Cell Cycle , Cell Proliferation/drug effects , Disease Models, Animal , Female , Gene Expression , Homeodomain Proteins/genetics , Malnutrition/metabolism , Metabolomics , Methionine/administration & dosage , Methionine/deficiency , Methionine/pharmacology , Mice , Mice, Transgenic , Nephrons/metabolism , Nephrons/pathology , Organ Culture Techniques , Pregnancy , RNA, Messenger , RNA-Seq , Signal Transduction , Stem Cells/physiology , Transcription Factors/genetics , Tuberous Sclerosis Complex 1 Protein/geneticsABSTRACT
There is a global epidemic of chronic kidney disease (CKD) characterized by a progressive loss of nephrons, ascribed in large part to a rising incidence of hypertension, metabolic syndrome, and type 2 diabetes mellitus. There is a ten-fold variation in nephron number at birth in the general population, and a 50% overall decrease in nephron number in the last decades of life. The vicious cycle of nephron loss stimulating hypertrophy by remaining nephrons and resulting in glomerulosclerosis has been regarded as maladaptive, and only partially responsive to angiotensin inhibition. Advances over the past century in kidney physiology, genetics, and development have elucidated many aspects of nephron formation, structure and function. Parallel advances have been achieved in evolutionary biology, with the emergence of evolutionary medicine, a discipline that promises to provide new insight into the treatment of chronic disease. This review provides a framework for understanding the origins of contemporary developmental nephrology, and recent progress in evolutionary biology. The establishment of evolutionary developmental biology (evo-devo), ecological developmental biology (eco-devo), and developmental origins of health and disease (DOHaD) followed the discovery of the hox gene family, the recognition of the contribution of cumulative environmental stressors to the changing phenotype over the life cycle, and mechanisms of epigenetic regulation. The maturation of evolutionary medicine has contributed to new investigative approaches to cardiovascular disease, cancer, and infectious disease, and promises the same for CKD. By incorporating these principles, developmental nephrology is ideally positioned to answer important questions regarding the fate of nephrons from embryo through senescence.
Subject(s)
Developmental Biology/methods , Evolution, Molecular , Nephrons/metabolism , Renal Insufficiency, Chronic/genetics , Animals , Developmental Biology/trends , Epigenesis, Genetic/genetics , Gene Expression Regulation, Developmental , Humans , Nephrons/cytology , Nephrons/embryology , Organogenesis/genetics , Renal Insufficiency, Chronic/embryology , Renal Insufficiency, Chronic/pathologyABSTRACT
Developmental changes in cell fate are tightly regulated by cell-type specific transcription factors. Chromatin reorganization during organismal development ensures dynamic access of developmental regulators to their cognate DNA sequences. Thus, understanding the epigenomic states of promoters and enhancers is of key importance. Recent years have witnessed significant advances in our knowledge of the transcriptional mechanisms of kidney development. Emerging evidence suggests that histone deacetylation by class I HDACs and H3 methylation on lysines 4, 27 and 79 play important roles in regulation of early and late gene expression in the developing kidney. Equally exciting is the realization that nephrogenesis genes in mesenchymal nephron progenitors harbor bivalent chromatin domains which resolve upon differentiation implicating chromatin bivalency in developmental control of gene expression. Here, we review current knowledge of the epigenomic states of nephric cells and current techniques used to study the dynamic chromatin states. These technological advances will provide an unprecedented view of the enhancer landscape during cell fate commitment and help in defining the complex transcriptional networks governing kidney development and disease.
Subject(s)
Epigenesis, Genetic , Epigenomics/methods , Kidney/metabolism , Nephrons/metabolism , Animals , Cell Differentiation/genetics , Gene Expression Regulation, Developmental , Humans , Kidney/cytology , Kidney/embryology , Kidney Diseases/embryology , Kidney Diseases/genetics , Kidney Diseases/pathology , Nephrons/cytology , Nephrons/embryology , Organogenesis/geneticsABSTRACT
The kidney vasculature has a unique and complex architecture that is central for the kidney to exert its multiple and essential physiological functions with the ultimate goal of maintaining homeostasis. An appropriate development and coordinated assembly of the different vascular cell types and their association with the corresponding nephrons is crucial for the generation of a functioning kidney. In this review we provide an overview of the renal vascular anatomy, histology, and current knowledge of the embryological origin and molecular pathways involved in its development. Understanding the cellular and molecular mechanisms involved in renal vascular development is the first step to advance the field of regenerative medicine.
Subject(s)
Kidney/blood supply , Neovascularization, Physiologic/physiology , Nephrons/blood supply , Renal Artery/anatomy & histology , Renal Veins/anatomy & histology , Animals , Gene Expression Regulation, Developmental , Humans , Kidney/embryology , Kidney/metabolism , Neovascularization, Physiologic/genetics , Nephrons/embryology , Nephrons/metabolism , Regenerative Medicine/methods , Regenerative Medicine/trends , Renal Artery/embryology , Renal Artery/metabolism , Renal Veins/embryology , Renal Veins/metabolismABSTRACT
Chromatin-remodeling complexes play critical roles in establishing gene expression patterns in response to developmental signals. How these epigenetic regulators determine the fate of progenitor cells during development of specific organs is not well understood. We found that genetic deletion of Brg1 (Smarca4), the core enzymatic protein in SWI/SNF, in nephron progenitor cells leads to severe renal hypoplasia. Nephron progenitor cells were depleted in Six2-Cre, Brg1flx/flx mice due to reduced cell proliferation. This defect in self-renewal, together with impaired differentiation resulted in a profound nephron deficit in Brg1 mutant kidneys. Sall1, a transcription factor that is required for expansion and maintenance of nephron progenitors, associates with SWI/SNF. Brg1 and Sall1 bind promoters of many progenitor cell genes and regulate expression of key targets that promote their proliferation.
Subject(s)
Cell Differentiation , Cell Proliferation , DNA Helicases/metabolism , Nephrons/embryology , Nuclear Proteins/metabolism , Stem Cells/metabolism , Transcription Factors/metabolism , Animals , COS Cells , Chlorocebus aethiops , DNA Helicases/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Mice , Mice, Transgenic , Nephrons/cytology , Nuclear Proteins/genetics , Stem Cells/cytology , Transcription Factors/geneticsABSTRACT
Epigenetic regulation of gene expression has a crucial role allowing for the self-renewal and differentiation of stem and progenitor populations during organogenesis. The mammalian kidney maintains a population of self-renewing stem cells that differentiate to give rise to thousands of nephrons, which are the functional units that carry out filtration to maintain physiological homeostasis. The polycomb repressive complex 2 (PRC2) epigenetically represses gene expression during development by placing the H3K27me3 mark on histone H3 at promoter and enhancer sites, resulting in gene silencing. To understand the role of PRC2 in nephron differentiation, we conditionally inactivated the Eed gene, which encodes a nonredundant component of the PRC2 complex, in nephron progenitor cells. Resultant kidneys were smaller and showed premature loss of progenitor cells. The progenitors in Eed mutant mice that were induced to differentiate did not develop into properly formed nephrons. Lhx1, normally expressed in the renal vesicle, was overexpressed in kidneys of Eed mutant mice. Thus, PRC2 has a crucial role in suppressing the expression of genes that maintain the progenitor state, allowing nephron differentiation to proceed.
Subject(s)
Cell Differentiation/physiology , Epigenesis, Genetic/physiology , Gene Expression Regulation, Developmental/physiology , Nephrons/embryology , Polycomb Repressive Complex 2/biosynthesis , Stem Cells/metabolism , Animals , LIM-Homeodomain Proteins/biosynthesis , LIM-Homeodomain Proteins/genetics , Mice , Mice, Transgenic , Mutation , Nephrons/cytology , Polycomb Repressive Complex 2/genetics , Stem Cells/cytology , Transcription Factors/biosynthesis , Transcription Factors/geneticsABSTRACT
Nephron progenitor cells (NPCs) are Six2-positive metanephric mesenchyme cells, which undergo self-renewal and differentiation to give rise to nephrons until the end of nephrogenesis. Histone deacetylases (HDACs) are a group of epigenetic regulators that control cell fate, but their role in balancing NPC renewal and differentiation is unknown. Here, we report that NPC-specific deletion of Hdac1 and Hdac2 genes in mice results in early postnatal lethality owing to renal hypodysplasia and loss of NPCs. HDAC1/2 interact with the NPC renewal regulators Six2, Osr1 and Sall1, and are co-bound along with Six2 on the Six2 enhancer. Although the mutant NPCs differentiate into renal vesicles (RVs), Hdac1/2 mutant kidneys lack nascent nephrons or mature glomeruli, a phenocopy of Lhx1 mutants. Transcriptional profiling and network analysis identified disrupted expression of Lhx1 and its downstream genes, Dll1 and Hnf1a/4a, as key mediators of the renal phenotype. Finally, although HDAC1/2-deficient NPCs and RVs overexpress hyperacetylated p53, Trp53 deletion failed to rescue the renal dysgenesis. We conclude that the epigenetic regulators HDAC1 and HDAC2 control nephrogenesis via interactions with the transcriptional programs of nephron progenitors and renal vesicles.
Subject(s)
Histone Deacetylase 1/genetics , Histone Deacetylase 2/genetics , Nephrons/embryology , Organogenesis/genetics , Stem Cells/cytology , Transcription, Genetic/genetics , Animals , Calcium-Binding Proteins , Cell Line , Cell Proliferation/genetics , HEK293 Cells , Hepatocyte Nuclear Factor 1-alpha/biosynthesis , Hepatocyte Nuclear Factor 4/biosynthesis , Histone Deacetylase 1/metabolism , Histone Deacetylase 2/metabolism , Homeodomain Proteins/metabolism , Humans , Intercellular Signaling Peptides and Proteins/biosynthesis , Kidney Diseases/genetics , LIM-Homeodomain Proteins/genetics , Mice , Mice, Knockout , Nephrons/cytology , Protein Serine-Threonine Kinases/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation/genetics , Tumor Suppressor Protein p53/geneticsABSTRACT
Normal kidney function depends on the proper development of the nephron: the functional unit of the kidney. Reciprocal signaling interactions between the stroma and nephron progenitor compartment have been proposed to control nephron development. Here, we show that removal of hedgehog intracellular effector smoothened (Smo-deficient mutants) in the cortical stroma results in an abnormal renal capsule, and an expanded nephron progenitor domain with an accompanying decrease in nephron number via a block in epithelialization. We show that stromal-hedgehog-Smo signaling acts through a GLI3 repressor. Whole-kidney RNA sequencing and analysis of FACS-isolated stromal cells identified impaired TGFß2 signaling in Smo-deficient mutants. We show that neutralization and knockdown of TGFß2 in explants inhibited nephrogenesis. In addition, we demonstrate that concurrent deletion of Tgfbr2 in stromal and nephrogenic cells in vivo results in decreased nephron formation and an expanded nephrogenic precursor domain similar to that observed in Smo-deficient mutant mice. Together, our data suggest a mechanism whereby a stromal hedgehog-TGFß2 signaling axis acts to control nephrogenesis.
Subject(s)
Forkhead Transcription Factors/metabolism , Hedgehog Proteins/metabolism , Nephrons/embryology , Signal Transduction/physiology , Smoothened Receptor/metabolism , Transforming Growth Factor beta2/metabolism , Animals , Forkhead Transcription Factors/genetics , Hedgehog Proteins/genetics , Mice , Mice, Knockout , Nephrons/cytology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Smoothened Receptor/genetics , Stromal Cells/cytology , Stromal Cells/metabolism , Transforming Growth Factor beta2/genetics , Zinc Finger Protein Gli3/genetics , Zinc Finger Protein Gli3/metabolismABSTRACT
The two major components of the metanephros, the urinary collecting system (UCS) and nephron, have different developmental courses. Nephron numbers vary widely between species and individuals and are determined during fetal development. Furthermore, the development of nascent nephrons may contribute to the expansion of the proximal part of the UCS. This study investigated the distribution of nascent nephrons and their interrelationship with UCS branches during human embryogenesis. We obtained samples from 31 human embryos between Carnegie stages (CSs) 19 and 23 from the Kyoto Collection at the Congenital Anomaly Research Center of Kyoto University in Japan. Serial histological sections of the metanephros with the UCS were digitalized and computationally reconstructed for morphological and quantitative analyses. The three-dimensional (3D) coordinates for the positions of all UCS branch points, end points, attachment points to nascent nephrons (APs), and renal corpuscles (RCs) were recorded and related to the developmental phase. Phases were categorized from phase 1 to phase 5 according to the histological analysis. The UCS branching continued until RCs first appeared (at CS19). End branches with attached nascent nephrons (EB-AP[+]) were observed after CS19 during the fifth generation or higher during the embryonic period. The range of end branch and EB-AP(+) generation numbers was broad, and the number of RCs increased with the embryonic stage, reaching 273.8 ± 104.2 at CS23. The number of RCs connected to the UCS exceeded the number not connected to the UCS by CS23. The 3D reconstructions revealed RCs to be distributed around end branches, close to the surface of the metanephros. The RCs connected to the UCS were located away from the surface. The APs remained near the end point, whereas connecting ducts that become renal tubules were found to elongate with maturation of the RCs. Nascent nephrons in RC phases 3-5 were preferentially attached to the end branches at CS22 and CS23. The mean generation number of EB-AP(-) was higher than that of EB-AP(+) in 19 of 22 metanephros and was statistically significant for eight metanephros at CS22 and CS23. The ratio of the deviated branching pattern was almost constant (29%). The ratio of the even branching pattern with EB-AP(+) and EB-AP(+) to the total even branching pattern increased with CS (9.2% at CS21, 19.2% at CS22, and 45.4% at CS23). Our data suggest the following: EB-AP(+) may not branch further at the tip (i.e., by tip splitting), but branching beneath the AP (lateral branching) continues throughout the embryonic stages. Our study provides valuable data that can further the understanding of the interactions between the UCS and nascent nephrons during human embryogenesis.
Subject(s)
Nephrons/embryology , Embryonic Development , HumansABSTRACT
Nephron formation continues throughout kidney morphogenesis in both mice and humans. Lineage tracing studies in mice identified a self-renewing Six2-expressing nephron progenitor population able to give rise to the full complement of nephrons throughout kidney morphogenesis. To investigate the origin of nephrons within human pluripotent stem cell-derived kidney organoids, we performed a similar fate-mapping analysis of the SIX2-expressing lineage in induced pluripotent stem cell (iPSC)-derived kidney organoids to explore the feasibility of investigating lineage relationships in differentiating iPSCs in vitro Using CRISPR/Cas9 gene-edited lineage reporter lines, we show that SIX2-expressing cells give rise to nephron epithelial cell types but not to presumptive ureteric epithelium. The use of an inducible (CreERT2) line revealed a declining capacity for SIX2+ cells to contribute to nephron formation over time, but retention of nephron-forming capacity if provided an exogenous WNT signal. Hence, while human iPSC-derived kidney tissue appears to maintain lineage relationships previously identified in developing mouse kidney, unlike the developing kidney in vivo, kidney organoids lack a nephron progenitor niche capable of both self-renewal and ongoing nephrogenesis.
Subject(s)
Chromosome Mapping , Gene Expression Profiling , Genes, Reporter , Nephrons/embryology , Nephrons/metabolism , Organogenesis/genetics , Biomarkers , CRISPR-Cas Systems , Cell Culture Techniques , Cell Differentiation , Homeodomain Proteins/genetics , Humans , Nerve Tissue Proteins/genetics , Organoids , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Single-Cell AnalysisABSTRACT
The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. Regenerating the kidney requires the induction of the more than 20 distinct cell types required for excretion and the regulation of pH, and electrolyte and fluid balance. We have previously described the simultaneous induction of progenitors for both collecting duct and nephrons via the directed differentiation of human pluripotent stem cells. Paradoxically, although both are of intermediate mesoderm in origin, collecting duct and nephrons have distinct temporospatial origins. Here we identify the developmental mechanism regulating the preferential induction of collecting duct versus kidney mesenchyme progenitors. Using this knowledge, we have generated kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells. Within these organoids, individual nephrons segment into distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization. When transcription profiles of kidney organoids were compared to human fetal tissues, they showed highest congruence with first trimester human kidney. Furthermore, the proximal tubules endocytose dextran and differentially apoptose in response to cisplatin, a nephrotoxicant. Such kidney organoids represent powerful models of the human organ for future applications, including nephrotoxicity screening, disease modelling and as a source of cells for therapy.
Subject(s)
Cell Lineage , Induced Pluripotent Stem Cells/cytology , Models, Biological , Nephrons/cytology , Nephrons/embryology , Organogenesis , Organoids/cytology , Animals , Coculture Techniques , Feeder Cells , Fetus/anatomy & histology , Fetus/cytology , Fetus/embryology , Fibroblasts/cytology , Humans , Kidney Tubules, Collecting/cytology , Kidney Tubules, Proximal/cytology , Kidney Tubules, Proximal/embryology , Kidney Tubules, Proximal/physiology , Mesoderm/cytology , Mice , Nephrons/anatomy & histology , Nephrons/physiology , Organoids/embryology , Tissue Culture TechniquesABSTRACT
Nephron progenitor number determines nephron endowment; a reduced nephron count is linked to the onset of kidney disease. Several transcriptional regulators including Six2, Wt1, Osr1, Sall1, Eya1, Pax2, and Hox11 paralogues are required for specification and/or maintenance of nephron progenitors. However, little is known about the regulatory intersection of these players. Here, we have mapped nephron progenitor-specific transcriptional networks of Six2, Hoxd11, Osr1, and Wt1. We identified 373 multi-factor associated 'regulatory hotspots' around genes closely associated with progenitor programs. To examine their functional significance, we deleted 'hotspot' enhancer elements for Six2 and Wnt4. Removal of the distal enhancer for Six2 leads to a ~40% reduction in Six2 expression. When combined with a Six2 null allele, progeny display a premature depletion of nephron progenitors. Loss of the Wnt4 enhancer led to a significant reduction of Wnt4 expression in renal vesicles and a mildly hypoplastic kidney, a phenotype also enhanced in combination with a Wnt4 null mutation. To explore the regulatory landscape that supports proper target gene expression, we performed CTCF ChIP-seq to identify insulator-boundary regions. One such putative boundary lies between the Six2 and Six3 loci. Evidence for the functional significance of this boundary was obtained by deep sequencing of the radiation-induced Brachyrrhine (Br) mutant allele. We identified an inversion of the Six2/Six3 locus around the CTCF-bound boundary, removing Six2 from its distal enhancer regulation, but placed next to Six3 enhancer elements which support ectopic Six2 expression in the lens where Six3 is normally expressed. Six3 is now predicted to fall under control of the Six2 distal enhancer. Consistent with this view, we observed ectopic Six3 in nephron progenitors. 4C-seq supports the model for Six2 distal enhancer interactions in wild-type and Br/+ mouse kidneys. Together, these data expand our view of the regulatory genome and regulatory landscape underpinning mammalian nephrogenesis.