Progenitor translatome changes coordinated by Tsc1 increase perception of Wnt signals to end nephrogenesis

AbstractMammalian nephron endowment is determined by the coordinated cessation of nephrogenesis in independent niches. Here we report that translatome analysis in Tsc1+/− nephron progenitor cells from mice with elevated nephron numbers reveals how differential translation of Wnt antagonists over agonists tips the balance between self-renewal and differentiation. Wnt agonists are poorly translated in young niches, resulting in an environment with low R-spondin and high Fgf20 promoting self-renewal. In older niches we find increased translation of Wnt agonists, including R-spondin and the signalosome-promoting Tmem59, and low Fgf20, promoting differentiation. This suggests that the tipping point for nephron progenitor exit from the niche is controlled by the gradual increase in stability and possibly clustering of Wnt/Fzd complexes in individual cells, enhancing the response to ureteric bud-derived Wnt9b inputs and driving synchronized differentiation. As predicted by these findings, removing one Rspo3 allele in nephron progenitors delays cessation and increases nephron numbers in vivo.

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Age-Dependent Changes in the Progenitor Translatome Coordinated in part by Tsc1 Increase Perception of Signaling Inputs to End Nephrogenesis

10.1101/2020.03.12.989343 ◽

2020 ◽

Author(s):

Eric Brunskill ◽

Alison Jarmas ◽

Praneet Chaturvedi ◽

Raphael Kopan

Keyword(s):

Gradual Increase ◽

Tipping Point ◽

Ureteric Bud ◽

Self Renewal ◽

Nephron Endowment ◽

Nephron Progenitors ◽

Age Dependent ◽

Cellular Translation ◽

Nephron Progenitor

AbstractMammalian nephron endowment is determined by the coordinated cessation of nephrogenesis in independent niches. Here we report that in young niches, cellular Wnt agonists are poorly translated, Fgf20 levels are high and R-spondin levels are low, resulting in a pro self-renewal environment. By contrast, older niches are low in Fgf20 and high in R-spondin, with increased cellular translation of Wnt agonists, including the signalosome-promoting Tmem59. This suggests a hypothesis that the tipping point for nephron progenitor exit from the niche is controlled by the gradual increase in stability and clustering of Wnt/Fzd complexes in individual cells, enhancing the response to ureteric bud-derived Wnt9b inputs and driving differentiation. We show Tsc1 hemizygosity differentially promoted translation of Wnt antagonists over agonists, expanding a transitional (Six2+, Cited1+, Wnt4+) state and delaying the tipping point. As predicted by these findings, reducing Rspo3 dosage in nephron progenitors or Tmem59 globally increased nephron numbers in vivo.

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Abstract 9: Histone Deacetylases 1 And 2 Regulate Six2 Function To Maintain Nephron Progenitor Cells During Nephrogenesis

Hypertension ◽

10.1161/hyp.76.suppl_1.9 ◽

2020 ◽

Vol 76 (Suppl_1) ◽

Author(s):

Hongbing Liu ◽

Mahitha M Koduri ◽

Andrea Dragon ◽

Chao Hui Chen ◽

Samir S El-Dahr

Keyword(s):

Progenitor Cells ◽

Histone Deacetylases ◽

Target Genes ◽

Fusion Gene ◽

Gene Expression Pattern ◽

Proximity Ligation Assay ◽

Self Renewal ◽

Nephron Endowment ◽

Nephron Progenitor

Low nephron endowment is strongly associated with cardiovascular disease, especially hypertension. Sine oculis homeobox 2 (Six2) is the master transcriptional regulator in balancing self-renewal and differentiation of nephron progenitor cells (NPCs) for appropriate nephron endowment. Loss of Six2 in mice causes early-onset loss of self-renewal and premature differentiation of NPCs. However, it is unclear how Six2 is functionally regulated during nephrogenesis. In vivo interaction of histone deacetylase1 and 2 (Hdac1/2) to Six2 was detected in developing kidney by co-immunoprecipitation and proximity ligation assay. Chromatin immunoprecipitation and DNA sequencing experiments in isolated E16.5 NPCs revealed 1,180 (84.58%) of the Six2 peaks overlapped with Hdac2 peaks, implying the involvement of Hdac1/2 in Six2 DNA binding and its function in NPCs. To test whether Hdac1/2 are required for Six2 function to regulate nephron formation, we employed Six2 GC mouse line, in which the eGFP and Cre fusion gene (GC) replaces and fully recapitulates the endogenous Six2 gene expression pattern. Analysis of kidneys at embryonic day (E) 19.5 and newborn (P0) showed that Six2 heterozygous (Six2 GC ) together with three alleles knockout of Hdac1/2 resulted in severely hypoplastic kidneys, while three alleles knockout of Hdac1/2 by transgenic Six2-Cre only led to very subtle phenotypes. Immunostaining at E 19.5 and P0 revealed about 50% reduction of Six2 level in the kidney of Six2 GC only and Six2 GC together with three alleles knockout of Hdac1/2 mice. In the kidneys of Six2 heterozygous mice, no change was observed for most of the NPC identity makers for self-renewal, including the three Six2 targets, Pax2, Sall1 and WT1. However, sequential removal of three alleles of Hdac1/2 of Six2 GC mice did not change Six2 protein level but significantly decreased the expression of Pax2, Sall1 and WT1, suggesting the requirement of Hdac1/2 for Six2’s function to transcriptionally activate the expression of its target genes. We also observed the premature differentiation and decreased nephron formation in mutant kidneys. Therefore, we conclude that Hdac1/2 are required for Six2’s function to promote NPC self-renewal and repress premature differentiation during nephrogenesis.

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Tumor-free elongation of mammalian nephrogenesis by excess fetal GDNF

10.1101/2020.05.28.120865 ◽

2020 ◽

Author(s):

Hao Li ◽

Jussi Kupari ◽

Yujuan Gui ◽

Edward Siefker ◽

Benson Lu ◽

...

Keyword(s):

Cardiovascular Health ◽

Major Effect ◽

Kidney Tumors ◽

Regenerative Capacity ◽

Self Renewal ◽

Fetal Period ◽

Nephron Endowment ◽

Nephron Progenitors ◽

Neurotropic Factor ◽

Nephron Progenitor

ABSTRACTDue to poor regenerative capacity of adult kidneys, nephron endowment defined by the nephrogenic program during the fetal period dictates renal and related cardiovascular health throughout life. We show that the neurotropic factor GDNF, which is in clinical trials for Parkinson’s disease, is capable of prolonging the nephrogenic program beyond its normal cessation without increasing the risk of kidney tumors. Our data demonstrates that excess GDNF expands the nephrogenic program by maintaining nephron progenitors and nephrogenesis in postnatal mouse kidneys. GDNF, through its transcriptional targets excreted from the adjacent epithelium, has a major effect on nephron progenitor self-renewal and maintenance; abnormally high GDNF inhibits nephron progenitor proliferation, but lowering its level normalizes the nephrogenic program to that permissive for nephron progenitor self-renewal and differentiation. Based on our results, we propose that the lifespan of nephron progenitors is determined by mechanisms related to perception of GDNF and other signaling levels.

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Wnt11 directs nephron progenitor polarity and motile behavior ultimately determining nephron endowment

eLife ◽

10.7554/elife.40392 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 11

Author(s):

Lori L O'Brien ◽

Alexander N Combes ◽

Kieran M Short ◽

Nils O Lindström ◽

Peter H Whitney ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cell ◽

Live Imaging ◽

Rna Seq ◽

Self Renewal ◽

Mammalian Kidney ◽

Nephron Endowment ◽

Nephron Progenitors ◽

Cell Organization ◽

Nephron Progenitor

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.

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Concurrent BMP7 and FGF9 signalling governs AP-1 function to promote self-renewal of nephron progenitor cells

Nature Communications ◽

10.1038/ncomms10027 ◽

2015 ◽

Vol 6 (1) ◽

Cited By ~ 31

Author(s):

Sree Deepthi Muthukrishnan ◽

Xuehui Yang ◽

Robert Friesel ◽

Leif Oxburgh

Keyword(s):

Progenitor Cells ◽

Self Renewal ◽

Nephron Progenitor

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The PI3K Pathway Balances Self-Renewal and Differentiation of Nephron Progenitor Cells through β-Catenin Signaling

Stem Cell Reports ◽

10.1016/j.stemcr.2015.01.021 ◽

2015 ◽

Vol 4 (4) ◽

pp. 551-560 ◽

Cited By ~ 19

Author(s):

Nils Olof Lindström ◽

Neil Oliver Carragher ◽

Peter Hohenstein

Keyword(s):

Progenitor Cells ◽

Pi3k Pathway ◽

Self Renewal ◽

Nephron Progenitor

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Cellular and molecular basis of haematopoiesis

Oxford Textbook of Medicine ◽

10.1093/med/9780198746690.003.0511 ◽

2020 ◽

pp. 5172-5181

Author(s):

Paresh Vyas ◽

N. Asger Jakobsen

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Cell Number ◽

Stages Of Development ◽

Self Renewal ◽

Haematopoietic System

Haematopoiesis involves a regulated set of developmental stages from haematopoietic stem cells (HSCs) that produce haematopoietic progenitor cells that then differentiate into more mature haematopoietic lineages, which provide all the key functions of the haematopoietic system. Definitive HSCs first develop within the embryo in specialized regions of the dorsal aorta and umbilical arteries and then seed the fetal liver and bone marrow. At the single-cell level, HSCs have the ability to reconstitute and maintain a functional haematopoietic system over extended periods of time in vivo. They (1) have a self-renewing capacity during the life of an organism, or even after transplantation; (2) are multipotent, with the ability to make all types of blood cells; and (3) are relatively quiescent, with the ability to serve as a deep reserve of cells to replenish short-lived, rapidly proliferation progenitors. Haematopoietic progenitor cells are unable to maintain long-term haematopoiesis in vivo due to limited or absent self-renewal. Rapid proliferation and cytokine responsiveness enables increased blood cell production under conditions of stress. Lineage commitment means limited cell type production. The haematopoietic stem cell niche is an anatomically and functionally defined regulatory environment for stem cells modulates self-renewal, differentiation, and proliferative activity of stem cells, thereby regulating stem cell number. Haematopoietic reconstitution during bone marrow transplantation is mediated by a succession of cells at various stages of development. More mature cells contribute to repopulation immediately following transplantation. With time, cells at progressively earlier stages of development are involved, with the final stable repopulation being provided by long-lived, multipotent HSCs. Long-term haematopoiesis is sustained by a relatively small number of HSCs.

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Three Optimized Methods for In Situ Quantification of Progenitor Cell Proliferation in Embryonic Kidneys Using BrdU, EdU, and PCNA

Canadian Journal of Kidney Health and Disease ◽

10.1177/2054358119871936 ◽

2019 ◽

Vol 6 ◽

pp. 205435811987193

Author(s):

Rosalie E. O’Hara ◽

Michel G. Arsenault ◽

Blanca P. Esparza Gonzalez ◽

Ashley Patriquen ◽

Sunny Hartwig

Keyword(s):

Cell Proliferation ◽

Progenitor Cells ◽

Kidney Development ◽

Fluorescence Labeling ◽

Nuclear Antigen ◽

Ureteric Bud ◽

Proliferating Cells ◽

Advantages And Disadvantages ◽

Nephron Progenitor

Background: Nephron progenitor cells derived from the metanephric mesenchyme undergo a complex balance of self-renewal and differentiation throughout kidney development to give rise to the mature nephron. Cell proliferation is an important index of progenitor population dynamics. However, accurate and reproducible in situ quantification of cell proliferation within progenitor populations can be technically difficult to achieve due to the complexity and harsh tissue treatment required of certain protocols. Objective: To optimize and compare the performance of the 3 most accurate S phase–specific labeling methods used for in situ detection and quantification of nephron progenitor and ureteric bud cell proliferation in the developing kidney, namely, 5-bromo-2’-deoxyuridine (BrdU), 5-ethynyl-2’-deoxyuridine (EdU), and proliferating cell nuclear antigen (PCNA). Methods: Protocols for BrdU, EdU, and PCNA were optimized for fluorescence labeling on paraformaldehyde-fixed, paraffin-embedded mouse kidney tissue sections, with co-labeling of nephron progenitor cells and ureteric bud with Six2 and E-cadherin antibodies, respectively. Image processing and analysis, including quantification of proliferating cells, were carried out using free ImageJ software. Results: All 3 methods detect similar ratios of nephron progenitor and ureteric bud proliferating cells. The BrdU staining protocol is the lengthiest and most complex protocol to perform, requires tissue denaturation, and is most subject to interexperimental signal variability. In contrast, bound PCNA and EdU protocols are relatively more straightforward, consistently yield clear results, and far more easily lend themselves to co-staining; however, the bound PCNA protocol requires substantive additional postexperimental analysis to distinguish the punctate nuclear PCNA staining pattern characteristic of proliferating cells. Conclusions: All 3 markers exhibit distinct advantages and disadvantages in quantifying cell proliferation in kidney progenitor populations, with EdU and PCNA protocols being favored due to greater technical ease and reproducibility of results associated with these methods.

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Chromatin Remodelers Interact with Eya1 and Six2 to Target Enhancers to Control Nephron Progenitor Cell Maintenance

Journal of the American Society of Nephrology ◽

10.1681/asn.2021040525 ◽

2021 ◽

Vol 32 (11) ◽

pp. 2815-2833

Author(s):

Jun Li ◽

Jinshu Xu ◽

Huihui Jiang ◽

Ting Zhang ◽

Aarthi Ramakrishnan ◽

...

Keyword(s):

Progenitor Cell ◽

Kidney Development ◽

Regulatory Elements ◽

Proximal Promoter ◽

Specific Expression ◽

Self Renewal ◽

Content Type ◽

Nephron Progenitors ◽

Genome Wide ◽

Nephron Progenitor

BackgroundEya1 is a critical regulator of nephron progenitor cell specification and interacts with Six2 to promote NPC self-renewal. Haploinsufficiency of these genes causes kidney hypoplasia. However, how the Eya1-centered network operates remains unknown.MethodsWe engineered a 2×HA-3×Flag-Eya1 knock-in mouse line and performed coimmunoprecipitation with anti-HA or -Flag to precipitate the multitagged-Eya1 and its associated proteins. Loss-of-function, transcriptome profiling, and genome-wide binding analyses for Eya1's interacting chromatin-remodeling ATPase Brg1 were carried out. We assayed the activity of the cis-regulatory elements co-occupied by Brg1/Six2 in vivo.ResultsEya1 and Six2 interact with the Brg1-based SWI/SNF complex during kidney development. Knockout of Brg1 results in failure of metanephric mesenchyme formation and depletion of nephron progenitors, which has been linked to loss of Eya1 expression. Transcriptional profiling shows conspicuous downregulation of important regulators for nephrogenesis in Brg1-deficient cells, including Lin28, Pbx1, and Dchs1-Fat4 signaling, but upregulation of podocyte lineage, oncogenic, and cell death–inducing genes, many of which Brg1 targets. Genome-wide binding analysis identifies Brg1 occupancy to a distal enhancer of Eya1 that drives nephron progenitor–specific expression. We demonstrate that Brg1 enrichment to two distal intronic enhancers of Pbx1 and a proximal promoter region of Mycn requires Six2 activity and that these Brg1/Six2-bound enhancers govern nephron progenitor–specific expression in response to Six2 activity.ConclusionsOur results reveal an essential role for Brg1, its downstream pathways, and its interaction with Eya1-Six2 in mediating the fine balance among the self-renewal, differentiation, and survival of nephron progenitors.

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