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Han C, Liu S, Ye S, Chen K, Chen D, Wang K, Liang W, Zhong S, Liu L, Li S, Chen W, Li Q. Genome-wide identification, evolution and expression of pax gene family members in mandarin fish (Siniperca chuatsi). COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY. PART D, GENOMICS & PROTEOMICS 2025; 54:101423. [PMID: 39842301 DOI: 10.1016/j.cbd.2025.101423] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/13/2024] [Revised: 12/31/2024] [Accepted: 01/16/2025] [Indexed: 01/24/2025]
Abstract
The pax gene family is involved in the development process through its extensive effects on cell proliferation, differentiation, and apoptosis. Herein, the whole pax gene family members of mandarin fish (Siniperca chuatsi) were first identified and characterized. By comparing pax gene family members from another 13 representative animals, an expansion of pax gene family members was observed in teleosts. In mandarin fish, a total of 15 potential pax gene family members, distributed on 13 chromosomes, were found, which shared conserved synteny with other teleosts. The expression profiles revealed that members of pax gene family showed time-specific expression profiles during embryonic and gonad development in mandarin fish, which indicated they might play a specific role in organogenesis during embryonic development and the process of gonad development and differentiation. Our research will lay a good foundation for further functional investigation of pax gene family during fish embryonic and gonad development.
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Affiliation(s)
- Chong Han
- Key Laboratory of Conservation and Application in Biodiversity of South China, School of Life Sciences, Guangzhou University, Guangzhou, 510006, China.
| | - Shiyan Liu
- State Key Laboratory of Biocontrol and School of Life Sciences, Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Guangdong Provincial Key Laboratory for Aquatic Economic Animals and Guangdong Provincial Engineering Technology Research Center for Healthy Breeding of Important Economic Fish, Sun Yat-Sen University, Guangzhou 510275, China
| | - Shuzheng Ye
- Key Laboratory of Conservation and Application in Biodiversity of South China, School of Life Sciences, Guangzhou University, Guangzhou, 510006, China
| | - Kaichun Chen
- Key Laboratory of Conservation and Application in Biodiversity of South China, School of Life Sciences, Guangzhou University, Guangzhou, 510006, China
| | - Dingxian Chen
- Key Laboratory of Conservation and Application in Biodiversity of South China, School of Life Sciences, Guangzhou University, Guangzhou, 510006, China
| | - Kaifeng Wang
- Key Laboratory of Conservation and Application in Biodiversity of South China, School of Life Sciences, Guangzhou University, Guangzhou, 510006, China
| | - Weiqian Liang
- Key Laboratory of Conservation and Application in Biodiversity of South China, School of Life Sciences, Guangzhou University, Guangzhou, 510006, China
| | - Simin Zhong
- Key Laboratory of Conservation and Application in Biodiversity of South China, School of Life Sciences, Guangzhou University, Guangzhou, 510006, China
| | - Lanyuan Liu
- Key Laboratory of Conservation and Application in Biodiversity of South China, School of Life Sciences, Guangzhou University, Guangzhou, 510006, China
| | - Sipeng Li
- Key Laboratory of Conservation and Application in Biodiversity of South China, School of Life Sciences, Guangzhou University, Guangzhou, 510006, China
| | - Weijian Chen
- Key Laboratory of Conservation and Application in Biodiversity of South China, School of Life Sciences, Guangzhou University, Guangzhou, 510006, China
| | - Qiang Li
- Key Laboratory of Conservation and Application in Biodiversity of South China, School of Life Sciences, Guangzhou University, Guangzhou, 510006, China.
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Achieng MA, Schnell J, Fausto CC, Csipán RL, Thornton ME, Grubbs BH, Lindström NO. Axial Nephron Fate Switching Demonstrates a Plastic System Tunable on Demand. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.03.29.646044. [PMID: 40236027 PMCID: PMC11996323 DOI: 10.1101/2025.03.29.646044] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/17/2025]
Abstract
The human nephron is a highly patterned tubular structure. It develops specialized cells that regulate bodily fluid homeostasis, blood pressure, and urine secretion throughout life. Approximately 1 million nephrons form in each kidney during embryonic and fetal development, but how they develop is poorly understood. Here we interrogate axial patterning mechanisms in the human nephron using an iPSC-derived kidney organoid system that generates hundreds of developmentally synchronized nephrons, and we compare it to in vivo human kidney development using single cell and spatial transcriptomic approaches. We show that human nephron patterning is controlled by integrated WNT/BMP/FGF signaling. Imposing a WNT ON /BMP OFF state established a distal nephron identity that matures into thick ascending loop of Henle cells by endogenously activating FGF. Simultaneous suppression of FGF signaling switches cells back to a proximal cell-state, a transformation that is in itself dependent on BMP signal transduction. Our system highlights plasticity in axial nephron patterning, delineates the roles of WNT, FGF, and BMP mediated mechanisms controlling nephron patterning, and paves the way for generating nephron cells on demand.
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Hu X, Sun J, Wan M, Zhang B, Wang L, Zhong TP. Expression levels and stoichiometry of Hnf1β, Emx2, Pax8 and Hnf4 influence direct reprogramming of induced renal tubular epithelial cells. CELL REGENERATION (LONDON, ENGLAND) 2024; 13:19. [PMID: 39347883 PMCID: PMC11442758 DOI: 10.1186/s13619-024-00202-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/11/2024] [Accepted: 09/18/2024] [Indexed: 10/01/2024]
Abstract
Generation of induced renal epithelial cells (iRECs) from fibroblasts offers great opportunities for renal disease modeling and kidney regeneration. However, the low reprogramming efficiency of the current approach to generate iRECs has hindered potential therapeutic application and regenerative approach. This could be in part attributed to heterogeneous and unbalanced expression of reprogramming factors (RFs) Hnf1β (H1), Emx2 (E), Pax8 (P), and Hnf4α (H4) in transduced fibroblasts. Here, we establish an advanced retroviral vector system that expresses H1, E, P, and H4 in high levels and distinct ratios from bicistronic transcripts separated by P2A. Mouse embryonic fibroblasts (MEFs) harboring Cdh16-Cre; mT/mG allele are utilized to conduct iREC reprogramming via directly monitoring single cell fate conversion. Three sets of bicistronic RF combinations including H1E/H4P, H1H4/EP, and H1P/H4E have been generated to induce iREC reprogramming. Each of the RF combinations gives rise to distinct H1, E, P, and H4 expression levels and different reprogramming efficiencies. The desired H1E/H4P combination that results in high expression levels of RFs with balanced stoichiometry. substantially enhances the efficiency and quality of iRECs compared with transduction of separate H1, E, P, and H4 lentiviruses. We find that H1E/H4P-induced iRECs exhibit the superior features of renal tubular epithelial cells, as evidenced by expressing renal tubular-specific genes, possessing endocytotic arrogation activity and assembling into tubules along decellularized kidney scaffolds. This study establishes H1E/H4P cassette as a valuable platform for future iREC studies and regenerative medicine.
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Affiliation(s)
- Xueli Hu
- Shanghai Key Laboratory of Regulatory Biology, Institute of Molecular Medicine, School of Life Sciences, East China Normal University, Shanghai, 200241, China
| | - Jianjian Sun
- Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, 510100, China
| | - Meng Wan
- Shanghai Key Laboratory of Regulatory Biology, Institute of Molecular Medicine, School of Life Sciences, East China Normal University, Shanghai, 200241, China
| | - Bianhong Zhang
- Shanghai Key Laboratory of Regulatory Biology, Institute of Molecular Medicine, School of Life Sciences, East China Normal University, Shanghai, 200241, China
| | - Linhui Wang
- Department of Urology, Changhai Hospital, Shanghai, 200433, China.
| | - Tao P Zhong
- Shanghai Key Laboratory of Regulatory Biology, Institute of Molecular Medicine, School of Life Sciences, East China Normal University, Shanghai, 200241, China.
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Schnell J, Miao Z, Achieng M, Fausto CC, Wang V, Kuyper FD, Thornton ME, Grubbs B, Kim J, Lindström NO. Stepwise developmental mimicry generates proximal-biased kidney organoids. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.06.28.601028. [PMID: 39005387 PMCID: PMC11244853 DOI: 10.1101/2024.06.28.601028] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/16/2024]
Abstract
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.
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Chambers BE, Weaver NE, Lara CM, Nguyen TK, Wingert RA. (Zebra)fishing for nephrogenesis genes. Tissue Barriers 2024; 12:2219605. [PMID: 37254823 PMCID: PMC11042071 DOI: 10.1080/21688370.2023.2219605] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2023] [Accepted: 05/14/2023] [Indexed: 06/01/2023] Open
Abstract
Kidney disease is a devastating condition affecting millions of people worldwide, where over 100,000 patients in the United States alone remain waiting for a lifesaving organ transplant. Concomitant with a surge in personalized medicine, single-gene mutations, and polygenic risk alleles have been brought to the forefront as core causes of a spectrum of renal disorders. With the increasing prevalence of kidney disease, it is imperative to make substantial strides in the field of kidney genetics. Nephrons, the core functional units of the kidney, are epithelial tubules that act as gatekeepers of body homeostasis by absorbing and secreting ions, water, and small molecules to filter the blood. Each nephron contains a series of proximal and distal segments with explicit metabolic functions. The embryonic zebrafish provides an ideal platform to systematically dissect the genetic cues governing kidney development. Here, we review the use of zebrafish to discover nephrogenesis genes.
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Affiliation(s)
- Brooke E. Chambers
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, Boler-Parseghian Center for Rare and Neglected Diseases, Warren Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana (IN), USA
| | - Nicole E. Weaver
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, Boler-Parseghian Center for Rare and Neglected Diseases, Warren Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana (IN), USA
| | - Caroline M. Lara
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, Boler-Parseghian Center for Rare and Neglected Diseases, Warren Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana (IN), USA
| | - Thanh Khoa Nguyen
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, Boler-Parseghian Center for Rare and Neglected Diseases, Warren Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana (IN), USA
| | - Rebecca A. Wingert
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, Boler-Parseghian Center for Rare and Neglected Diseases, Warren Center for Drug Discovery, University of Notre Dame, Notre Dame, Indiana (IN), USA
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6
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Zhang G, Fu Y, Yang L, Ye F, Zhang P, Zhang S, Ma L, Li J, Wu H, Han X, Wang J, Guo G. Construction of single-cell cross-species chromatin accessibility landscapes with combinatorial-hybridization-based ATAC-seq. Dev Cell 2024; 59:793-811.e8. [PMID: 38330939 DOI: 10.1016/j.devcel.2024.01.015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2023] [Revised: 11/03/2023] [Accepted: 01/18/2024] [Indexed: 02/10/2024]
Abstract
Despite recent advances in single-cell genomics, the lack of maps for single-cell candidate cis-regulatory elements (cCREs) in non-mammal species has limited our exploration of conserved regulatory programs across vertebrates and invertebrates. Here, we developed a combinatorial-hybridization-based method for single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) named CH-ATAC-seq, enabling the construction of single-cell accessible chromatin landscapes for zebrafish, Drosophila, and earthworms (Eisenia andrei). By integrating scATAC censuses of humans, monkeys, and mice, we systematically identified 152 distinct main cell types and around 0.8 million cell-type-specific cCREs. Our analysis provided insights into the conservation of neural, muscle, and immune lineages across species, while epithelial cells exhibited a higher organ-origin heterogeneity. Additionally, a large-scale gene regulatory network (GRN) was constructed in four vertebrates by integrating scRNA-seq censuses. Overall, our study provides a valuable resource for comparative epigenomics, identifying the evolutionary conservation and divergence of gene regulation across different species.
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Affiliation(s)
- Guodong Zhang
- Bone Marrow Transplantation Center of the First Affiliated Hospital, and Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou 310000, China; Liangzhu Laboratory, Zhejiang University, Hangzhou 311121, China
| | - Yuting Fu
- Bone Marrow Transplantation Center of the First Affiliated Hospital, and Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou 310000, China
| | - Lei Yang
- Bone Marrow Transplantation Center of the First Affiliated Hospital, and Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou 310000, China
| | - Fang Ye
- Bone Marrow Transplantation Center of the First Affiliated Hospital, and Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou 310000, China; Liangzhu Laboratory, Zhejiang University, Hangzhou 311121, China
| | - Peijing Zhang
- Bone Marrow Transplantation Center of the First Affiliated Hospital, and Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou 310000, China
| | - Shuang Zhang
- Bone Marrow Transplantation Center of the First Affiliated Hospital, and Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou 310000, China
| | - Lifeng Ma
- Bone Marrow Transplantation Center of the First Affiliated Hospital, and Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou 310000, China
| | - Jiaqi Li
- Bone Marrow Transplantation Center of the First Affiliated Hospital, and Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou 310000, China
| | - Hanyu Wu
- Bone Marrow Transplantation Center of the First Affiliated Hospital, and Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou 310000, China
| | - Xiaoping Han
- Bone Marrow Transplantation Center of the First Affiliated Hospital, and Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou 310000, China; Zhejiang Provincial Key Laboratory for Tissue Engineering and Regenerative Medicine, Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cell and Regenerative Medicine, Hangzhou 310058, China.
| | - Jingjing Wang
- Bone Marrow Transplantation Center of the First Affiliated Hospital, and Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou 310000, China; Liangzhu Laboratory, Zhejiang University, Hangzhou 311121, China.
| | - Guoji Guo
- Bone Marrow Transplantation Center of the First Affiliated Hospital, and Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou 310000, China; Liangzhu Laboratory, Zhejiang University, Hangzhou 311121, China; Zhejiang Provincial Key Laboratory for Tissue Engineering and Regenerative Medicine, Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cell and Regenerative Medicine, Hangzhou 310058, China; Institute of Hematology, Zhejiang University, Hangzhou, China.
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7
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Bahrami M, Darabi S, Roozbahany NA, Abbaszadeh HA, Moghadasali R. Great potential of renal progenitor cells in kidney: From the development to clinic. Exp Cell Res 2024; 434:113875. [PMID: 38092345 DOI: 10.1016/j.yexcr.2023.113875] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2023] [Revised: 12/02/2023] [Accepted: 12/03/2023] [Indexed: 12/23/2023]
Abstract
The mammalian renal organ represents a pinnacle of complexity, housing functional filtering units known as nephrons. During embryogenesis, the depletion of niches containing renal progenitor cells (RPCs) and the subsequent incapacity of adult kidneys to generate new nephrons have prompted the formulation of protocols aimed at isolating residual RPCs from mature kidneys and inducing their generation from diverse cell sources, notably pluripotent stem cells. Recent strides in the realm of regenerative medicine and the repair of tissues using stem cells have unveiled critical signaling pathways essential for the maintenance and generation of human RPCs in vitro. These findings have ushered in a new era for exploring novel strategies for renal protection. The present investigation delves into potential transcription factors and signaling cascades implicated in the realm of renal progenitor cells, focusing on their protection and differentiation. The discourse herein elucidates contemporary research endeavors dedicated to the acquisition of progenitor cells, offering crucial insights into the developmental mechanisms of these cells within the renal milieu and paving the way for the formulation of innovative treatment modalities.
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Affiliation(s)
- Maryam Bahrami
- Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Laser Applications in Medical Sciences Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Shahram Darabi
- Cellular and Molecular Research Center, Research Institute for Non-Communicable Diseases, Qazvin University of Medical Sciences, Qazvin, Iran
| | | | - Hojjat Allah Abbaszadeh
- Laser Applications in Medical Sciences Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
| | - Reza Moghadasali
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
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8
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Nauryzgaliyeva Z, Goux Corredera I, Garreta E, Montserrat N. Harnessing mechanobiology for kidney organoid research. Front Cell Dev Biol 2023; 11:1273923. [PMID: 38077999 PMCID: PMC10704179 DOI: 10.3389/fcell.2023.1273923] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2023] [Accepted: 10/16/2023] [Indexed: 10/16/2024] Open
Abstract
Recently, organoids have emerged as revolutionizing tools with the unprecedented potential to recreate organ-specific microanatomy in vitro. Upon their derivation from human pluripotent stem cells (hPSCs), organoids reveal the blueprints of human organogenesis, further allowing the faithful recapitulation of their physiology. Nevertheless, along with the evolution of this field, advanced research exposed the organoids' shortcomings, particularly regarding poor reproducibility rates and overall immatureness. To resolve these challenges, many studies have started to underscore the relevance of mechanical cues as a relevant source to induce and externally control hPSCs differentiation. Indeed, established organoid generation protocols from hPSCs have mainly relyed on the biochemical induction of fundamental signalling pathways present during kidney formation in mammals, whereas mechanical cues have largely been unexplored. This review aims to discuss the pertinence of (bio) physical cues within hPSCs-derived organoid cultures, while deciphering their effect on morphogenesis. Moreover, we will explore state-of-the-art mechanobiology techniques as revolutionizing means for understanding the underlying role of mechanical forces in biological processes in organoid model systems.
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Affiliation(s)
- Zarina Nauryzgaliyeva
- Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain
| | - Iphigénie Goux Corredera
- Pluripotency for Organ Regeneration, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), University of Barcelona, Barcelona, Spain
| | - Elena Garreta
- Pluripotency for Organ Regeneration, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), University of Barcelona, Barcelona, Spain
| | - Nuria Montserrat
- Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain
- Pluripotency for Organ Regeneration, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), University of Barcelona, Barcelona, Spain
- Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina, Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain
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9
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Li L, Li CG, Almomani SN, Hossain SM, Eccles MR. Co-Expression of Multiple PAX Genes in Renal Cell Carcinoma (RCC) and Correlation of High PAX Expression with Favorable Clinical Outcome in RCC Patients. Int J Mol Sci 2023; 24:11432. [PMID: 37511191 PMCID: PMC10380508 DOI: 10.3390/ijms241411432] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2023] [Revised: 07/10/2023] [Accepted: 07/11/2023] [Indexed: 07/30/2023] Open
Abstract
Renal cell carcinoma (RCC) is the most common form of kidney cancer, consisting of multiple distinct subtypes. RCC has the highest mortality rate amongst the urogenital cancers, with kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), and kidney chromophobe carcinoma (KICH) being the most common subtypes. The Paired-box (PAX) gene family encodes transcription factors, which orchestrate multiple processes in cell lineage determination during embryonic development and organogenesis. Several PAX genes have been shown to be expressed in RCC following its onset and progression. Here, we performed real-time quantitative polymerase chain reaction (RT-qPCR) analysis on a series of human RCC cell lines, revealing significant co-expression of PAX2, PAX6, and PAX8. Knockdown of PAX2 or PAX8 mRNA expression using RNA interference (RNAi) in the A498 RCC cell line resulted in inhibition of cell proliferation, which aligns with our previous research, although no reduction in cell proliferation was observed using a PAX2 small interfering RNA (siRNA). We downloaded publicly available RNA-sequencing data and clinical histories of RCC patients from The Cancer Genome Atlas (TCGA) database. Based on the expression levels of PAX2, PAX6, and PAX8, RCC patients were categorized into two PAX expression subtypes, PAXClusterA and PAXClusterB, exhibiting significant differences in clinical characteristics. We found that the PAXClusterA expression subgroup was associated with favorable clinical outcomes and better overall survival. These findings provide novel insights into the association between PAX gene expression levels and clinical outcomes in RCC patients, potentially contributing to improved treatment strategies for RCC.
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Affiliation(s)
- Lei Li
- Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin 9016, New Zealand
| | - Caiyun G Li
- Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Suzan N Almomani
- Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin 9016, New Zealand
- Maurice Wilkins Centre for Molecular Biodiscovery, Level 2, 3A Symonds Street, Auckland 1010, New Zealand
| | - Sultana Mehbuba Hossain
- Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin 9016, New Zealand
- Maurice Wilkins Centre for Molecular Biodiscovery, Level 2, 3A Symonds Street, Auckland 1010, New Zealand
| | - Michael R Eccles
- Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin 9016, New Zealand
- Maurice Wilkins Centre for Molecular Biodiscovery, Level 2, 3A Symonds Street, Auckland 1010, New Zealand
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10
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Nguyen TK, Petrikas M, Chambers BE, Wingert RA. Principles of Zebrafish Nephron Segment Development. J Dev Biol 2023; 11:jdb11010014. [PMID: 36976103 PMCID: PMC10052950 DOI: 10.3390/jdb11010014] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2023] [Revised: 03/08/2023] [Accepted: 03/15/2023] [Indexed: 03/29/2023] Open
Abstract
Nephrons are the functional units which comprise the kidney. Each nephron contains a number of physiologically unique populations of specialized epithelial cells that are organized into discrete domains known as segments. The principles of nephron segment development have been the subject of many studies in recent years. Understanding the mechanisms of nephrogenesis has enormous potential to expand our knowledge about the basis of congenital anomalies of the kidney and urinary tract (CAKUT), and to contribute to ongoing regenerative medicine efforts aimed at identifying renal repair mechanisms and generating replacement kidney tissue. The study of the zebrafish embryonic kidney, or pronephros, provides many opportunities to identify the genes and signaling pathways that control nephron segment development. Here, we describe recent advances of nephron segment patterning and differentiation in the zebrafish, with a focus on distal segment formation.
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Affiliation(s)
- Thanh Khoa Nguyen
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, Boler-Parseghian Center for Rare and Neglected Diseases, Warren Center for Drug Discovery, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Madeline Petrikas
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, Boler-Parseghian Center for Rare and Neglected Diseases, Warren Center for Drug Discovery, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Brooke E Chambers
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, Boler-Parseghian Center for Rare and Neglected Diseases, Warren Center for Drug Discovery, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Rebecca A Wingert
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, Boler-Parseghian Center for Rare and Neglected Diseases, Warren Center for Drug Discovery, University of Notre Dame, Notre Dame, IN 46556, USA
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11
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Drummond BE, Ercanbrack WS, Wingert RA. Modeling Podocyte Ontogeny and Podocytopathies with the Zebrafish. J Dev Biol 2023; 11:9. [PMID: 36810461 PMCID: PMC9944608 DOI: 10.3390/jdb11010009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2023] [Revised: 02/11/2023] [Accepted: 02/17/2023] [Indexed: 02/22/2023] Open
Abstract
Podocytes are exquisitely fashioned kidney cells that serve an essential role in the process of blood filtration. Congenital malformation or damage to podocytes has dire consequences and initiates a cascade of pathological changes leading to renal disease states known as podocytopathies. In addition, animal models have been integral to discovering the molecular pathways that direct the development of podocytes. In this review, we explore how researchers have used the zebrafish to illuminate new insights about the processes of podocyte ontogeny, model podocytopathies, and create opportunities to discover future therapies.
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Affiliation(s)
| | | | - Rebecca A. Wingert
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, Boler-Parseghian Center for Rare and Neglected Diseases, Warren Center for Drug Discovery, University of Notre Dame, Notre Dame, IN 46556, USA
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12
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Wesselman HM, Gatz AE, Pfaff MR, Arceri L, Wingert RA. Estrogen Signaling Influences Nephron Segmentation of the Zebrafish Embryonic Kidney. Cells 2023; 12:666. [PMID: 36831333 PMCID: PMC9955091 DOI: 10.3390/cells12040666] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2023] [Revised: 02/13/2023] [Accepted: 02/16/2023] [Indexed: 02/22/2023] Open
Abstract
Despite significant advances in understanding nephron segment patterning, many questions remain about the underlying genes and signaling pathways that orchestrate renal progenitor cell fate choices and regulate differentiation. In an effort to identify elusive regulators of nephron segmentation, our lab conducted a high-throughput drug screen using a bioactive chemical library and developing zebrafish, which are a conserved vertebrate model and particularly conducive to large-scale screening approaches. 17β-estradiol (E2), which is the dominant form of estrogen in vertebrates, was a particularly interesting hit from this screen. E2 has been extensively studied in the context of gonad development, but roles for E2 in nephron development were unknown. Here, we report that exogenous estrogen treatments affect distal tubule composition, namely, causing an increase in the distal early segment and a decrease in the neighboring distal late. These changes were noted early in development but were not due to changes in cell dynamics. Interestingly, exposure to the xenoestrogens ethinylestradiol and genistein yielded the same changes in distal segments. Further, upon treatment with an estrogen receptor 2 (Esr2) antagonist, PHTPP, we observed the opposite phenotypes. Similarly, genetic deficiency of the Esr2 analog, esr2b, revealed phenotypes consistent with that of PHTPP treatment. Inhibition of E2 signaling also resulted in decreased expression of essential distal transcription factors, irx3b and its target irx1a. These data suggest that estrogenic compounds are essential for distal segment fate during nephrogenesis in the zebrafish pronephros and expand our fundamental understanding of hormone function during kidney organogenesis.
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Affiliation(s)
| | | | | | | | - Rebecca A. Wingert
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, Boler-Parseghian Center for Rare and Neglected Diseases, Warren Center for Drug Discovery, University of Notre Dame, Notre Dame, IN 46556, USA
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13
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Wesselman HM, Gatz AE, Wingert RA. Visualizing multiciliated cells in the zebrafish. Methods Cell Biol 2023; 175:129-161. [PMID: 36967138 DOI: 10.1016/bs.mcb.2022.12.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Ciliated cells serve vital functions in the body ranging from mechano- and chemo-sensing to fluid propulsion. Specialized cells with bundles dozens to hundreds of motile cilia known as multiciliated cells (MCCs) are essential as well, where they direct fluid movement in locations such as the respiratory, central nervous and reproductive systems. Intriguingly, the appearance of MCCs has been noted in the kidney in several disease conditions, but knowledge about their contributions to the pathobiology of these states has remained a mystery. As the mechanisms contributing to ciliopathic diseases are not yet fully understood, animal models serve as valuable tools for studying cilia development and how alterations in ciliated cell function impacts disease progression. Like other vertebrates, the zebrafish, Danio rerio, has numerous ciliated tissues. Among these, the embryonic kidney (or pronephros) is comprised of both monociliated cells and MCCs and therefore provides a setting to investigate both ciliated cell fate choice and ciliogenesis. Considering the zebrafish nephron resembles the segmentation and function of human nephrons, the zebrafish provide a tractable model for studying conserved ciliogenesis pathways in vivo. In this chapter, we provide an overview of ciliated cells with a special focus on MCCs, and present a suite of methods that can be used to visualize ciliated cells and their features in the developing zebrafish. Further, these methods enable precise quantification of ciliated cell number and various cilia-related characteristics.
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14
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Weaver NE, Healy A, Wingert RA. gldc Is Essential for Renal Progenitor Patterning during Kidney Development. Biomedicines 2022; 10:biomedicines10123220. [PMID: 36551976 PMCID: PMC9776136 DOI: 10.3390/biomedicines10123220] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2022] [Revised: 12/04/2022] [Accepted: 12/07/2022] [Indexed: 12/14/2022] Open
Abstract
The glycine cleavage system (GCS) is a complex located on the mitochondrial membrane that is responsible for regulating glycine levels and contributing one-carbon units to folate metabolism. Congenital mutations in GCS components, such as glycine decarboxylase (gldc), cause an elevation in glycine levels and the rare disease, nonketotic hyperglycinemia (NKH). NKH patients suffer from pleiotropic symptoms including seizures, lethargy, mental retardation, and early death. Therefore, it is imperative to fully elucidate the pathological effects of gldc dysfunction and glycine accumulation during development. Here, we describe a zebrafish model of gldc deficiency that recapitulates phenotypes seen in humans and mice. gldc deficient embryos displayed impaired fluid homeostasis suggesting renal abnormalities, as well as aberrant craniofacial morphology and neural development defects. Whole mount in situ hybridization (WISH) revealed that gldc transcripts were highly expressed in the embryonic kidney, as seen in mouse and human repository data, and that formation of several nephron segments was disrupted in gldc deficient embryos, including proximal and distal tubule populations. These kidney defects were caused by alterations in renal progenitor populations, revealing that the proper function of Gldc is essential for the patterning of this organ. Additionally, further analysis of the urogenital tract revealed altered collecting duct and cloaca morphology in gldc deficient embryos. Finally, to gain insight into the molecular mechanisms underlying these disruptions, we examined the effects of exogenous glycine treatment and observed analogous renal and cloacal defects. Taken together, these studies indicate for the first time that gldc function serves an essential role in regulating renal progenitor development by modulating glycine levels.
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15
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Hnf1b renal expression directed by a distal enhancer responsive to Pax8. Sci Rep 2022; 12:19921. [PMID: 36402859 PMCID: PMC9675860 DOI: 10.1038/s41598-022-21171-x] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2022] [Accepted: 09/23/2022] [Indexed: 11/21/2022] Open
Abstract
Xenopus provides a simple and efficient model system to study nephrogenesis and explore the mechanisms causing renal developmental defects in human. Hnf1b (hepatocyte nuclear factor 1 homeobox b), a gene whose mutations are the most commonly identified genetic cause of developmental kidney disease, is required for the acquisition of a proximo-intermediate nephron segment in Xenopus as well as in mouse. Genetic networks involved in Hnf1b expression during kidney development remain poorly understood. We decided to explore the transcriptional regulation of Hnf1b in the developing Xenopus pronephros and mammalian renal cells. Using phylogenetic footprinting, we identified an evolutionary conserved sequence (CNS1) located several kilobases (kb) upstream the Hnf1b transcription start and harboring epigenomic marks characteristics of a distal enhancer in embryonic and adult renal cells in mammals. By means of functional expression assays in Xenopus and mammalian renal cell lines we showed that CNS1 displays enhancer activity in renal tissue. Using CRISPR/cas9 editing in Xenopus tropicalis, we demonstrated the in vivo functional relevance of CNS1 in driving hnf1b expression in the pronephros. We further showed the importance of Pax8-CNS1 interaction for CNS1 enhancer activity allowing us to conclude that Hnf1b is a direct target of Pax8. Our work identified for the first time a Hnf1b renal specific enhancer and may open important perspectives into the diagnosis for congenital kidney anomalies in human, as well as modeling HNF1B-related diseases.
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16
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Drummond BE, Chambers BE, Wesselman HM, Gibson S, Arceri L, Ulrich MN, Gerlach GF, Kroeger PT, Leshchiner I, Goessling W, Wingert RA. osr1 Maintains Renal Progenitors and Regulates Podocyte Development by Promoting wnt2ba via the Antagonism of hand2. Biomedicines 2022; 10:biomedicines10112868. [PMID: 36359386 PMCID: PMC9687957 DOI: 10.3390/biomedicines10112868] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2022] [Revised: 11/01/2022] [Accepted: 11/05/2022] [Indexed: 11/11/2022] Open
Abstract
Knowledge about the genetic pathways that control nephron development is essential for better understanding the basis of congenital malformations of the kidney. The transcription factors Osr1 and Hand2 are known to exert antagonistic influences to balance kidney specification. Here, we performed a forward genetic screen to identify nephrogenesis regulators, where whole genome sequencing identified an osr1 lesion in the novel oceanside (ocn) mutant. The characterization of the mutant revealed that osr1 is needed to specify not renal progenitors but rather their maintenance. Additionally, osr1 promotes the expression of wnt2ba in the intermediate mesoderm (IM) and later the podocyte lineage. wnt2ba deficiency reduced podocytes, where overexpression of wnt2ba was sufficient to rescue podocytes and osr1 deficiency. Antagonism between osr1 and hand2 mediates podocyte development specifically by controlling wnt2ba expression. These studies reveal new insights about the roles of Osr1 in promoting renal progenitor survival and lineage choice.
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Affiliation(s)
- Bridgette E. Drummond
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Brooke E. Chambers
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Hannah M. Wesselman
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Shannon Gibson
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Liana Arceri
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Marisa N. Ulrich
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Gary F. Gerlach
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Paul T. Kroeger
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Ignaty Leshchiner
- Brigham and Women’s Hospital, Genetics and Gastroenterology Division, Harvard Medical School, Harvard Stem Cell Institute, Boston, MA 02215, USA
| | - Wolfram Goessling
- Brigham and Women’s Hospital, Genetics and Gastroenterology Division, Harvard Medical School, Harvard Stem Cell Institute, Boston, MA 02215, USA
| | - Rebecca A. Wingert
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
- Brigham and Women’s Hospital, Genetics and Gastroenterology Division, Harvard Medical School, Harvard Stem Cell Institute, Boston, MA 02215, USA
- Correspondence: ; Tel.: +1-574-631-0907
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17
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Mattonet K, Riemslagh FW, Guenther S, Prummel KD, Kesavan G, Hans S, Ebersberger I, Brand M, Burger A, Reischauer S, Mosimann C, Stainier DYR. Endothelial versus pronephron fate decision is modulated by the transcription factors Cloche/Npas4l, Tal1, and Lmo2. SCIENCE ADVANCES 2022; 8:eabn2082. [PMID: 36044573 PMCID: PMC9432843 DOI: 10.1126/sciadv.abn2082] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/10/2021] [Accepted: 07/11/2022] [Indexed: 05/17/2023]
Abstract
Endothelial specification is a key event during embryogenesis; however, when, and how, endothelial cells separate from other lineages is poorly understood. In zebrafish, Npas4l is indispensable for endothelial specification by inducing the expression of the transcription factor genes etsrp, tal1, and lmo2. We generated a knock-in reporter in zebrafish npas4l to visualize endothelial progenitors and their derivatives in wild-type and mutant embryos. Unexpectedly, we find that in npas4l mutants, npas4l reporter-expressing cells contribute to the pronephron tubules. Single-cell transcriptomics and live imaging of the early lateral plate mesoderm in wild-type embryos indeed reveals coexpression of endothelial and pronephron markers, a finding confirmed by creERT2-based lineage tracing. Increased contribution of npas4l reporter-expressing cells to pronephron tubules is also observed in tal1 and lmo2 mutants and is reversed in npas4l mutants injected with tal1 mRNA. Together, these data reveal that Npas4l/Tal1/Lmo2 regulate the fate decision between the endothelial and pronephron lineages.
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Affiliation(s)
- Kenny Mattonet
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, 61231, Germany
- DZHK (German Center for Cardiovascular Research), partner site, 43, D-61231 Bad Nauheim
- CPI (Cardio Pulmonary Institute), partner site, 43, D-61231 Bad Nauheim
- DZL (German Center for Lung Research), partner site, 43, D-61231 Bad Nauheim
| | - Fréderike W. Riemslagh
- Section of Developmental Biology, Department of Pediatrics, University of Colorado School of Medicine, Anschutz Medical Campus, 12801 E 17th Avenue, Aurora, CO 80045, USA
| | - Stefan Guenther
- DZHK (German Center for Cardiovascular Research), partner site, 43, D-61231 Bad Nauheim
- CPI (Cardio Pulmonary Institute), partner site, 43, D-61231 Bad Nauheim
- Bioinformatics and Deep Sequencing Platform, Max Planck Institute for Heart and Lung Research, Bad Nauheim 61231, Germany
| | - Karin D. Prummel
- Section of Developmental Biology, Department of Pediatrics, University of Colorado School of Medicine, Anschutz Medical Campus, 12801 E 17th Avenue, Aurora, CO 80045, USA
| | - Gokul Kesavan
- Center for Regenerative Therapies at TU Dresden (CRTD); Dresden, Germany
| | - Stefan Hans
- Center for Regenerative Therapies at TU Dresden (CRTD); Dresden, Germany
| | - Ingo Ebersberger
- Goethe University Frankfurt am Main, Institute of Cell Biology and Neuroscience, Frankfurt 60438, Germany
- Senckenberg Biodiversity and Climate Research Center (S-BIKF), Frankfurt 60325, Germany
- LOEWE Center for Translational Biodiversity Genomics (TBG), Frankfurt 60325, Germany
| | - Michael Brand
- Center for Regenerative Therapies at TU Dresden (CRTD); Dresden, Germany
| | - Alexa Burger
- Section of Developmental Biology, Department of Pediatrics, University of Colorado School of Medicine, Anschutz Medical Campus, 12801 E 17th Avenue, Aurora, CO 80045, USA
| | - Sven Reischauer
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, 61231, Germany
- CPI (Cardio Pulmonary Institute), partner site, 43, D-61231 Bad Nauheim
| | - Christian Mosimann
- Section of Developmental Biology, Department of Pediatrics, University of Colorado School of Medicine, Anschutz Medical Campus, 12801 E 17th Avenue, Aurora, CO 80045, USA
| | - Didier Y. R. Stainier
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, 61231, Germany
- DZHK (German Center for Cardiovascular Research), partner site, 43, D-61231 Bad Nauheim
- CPI (Cardio Pulmonary Institute), partner site, 43, D-61231 Bad Nauheim
- DZL (German Center for Lung Research), partner site, 43, D-61231 Bad Nauheim
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18
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Tran T, Song CJ, Nguyen T, Cheng SY, McMahon JA, Yang R, Guo Q, Der B, Lindström NO, Lin DCH, McMahon AP. A scalable organoid model of human autosomal dominant polycystic kidney disease for disease mechanism and drug discovery. Cell Stem Cell 2022; 29:1083-1101.e7. [PMID: 35803227 PMCID: PMC11088748 DOI: 10.1016/j.stem.2022.06.005] [Citation(s) in RCA: 71] [Impact Index Per Article: 23.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2020] [Revised: 01/28/2022] [Accepted: 06/08/2022] [Indexed: 12/13/2022]
Abstract
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.
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Affiliation(s)
- Tracy Tran
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Cheng Jack Song
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA; Amgen Research, Cardiometabolic Disorders, 1120 Veterans Blvd, South San Francisco, CA 94080, USA
| | - Trang Nguyen
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Shun-Yang Cheng
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Jill A McMahon
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Rui Yang
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Qiuyu Guo
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Balint Der
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Nils O Lindström
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Daniel C-H Lin
- Amgen Research, Cardiometabolic Disorders, 1120 Veterans Blvd, South San Francisco, CA 94080, USA
| | - Andrew P McMahon
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA.
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19
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Li JT, Zhang YD, Song XR, Li RJ, Yang WL, Tian M, Zhang SF, Cao GH, Song LL, Chen YM, Liu CH. The mechanism and effects of remdesivir-induced developmental toxicity in zebrafish: Blood flow dysfunction and behavioral alterations. J Appl Toxicol 2022; 42:1688-1700. [PMID: 35560222 DOI: 10.1002/jat.4336] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2021] [Revised: 04/01/2022] [Accepted: 04/30/2022] [Indexed: 11/11/2022]
Abstract
The antiviral drug remdesivir has been used to treat the growing number of coronavirus disease 2019 (COVID-19) patients. However, the drug is mainly excreted through urine and feces and introduced into the environment to affect non-target organisms, including fish, which has raised concerns about potential ecotoxicological effects on aquatic organisms. Moreover, studies on the ecological impacts of remdesivir on aquatic environments have not been reported. Here, we aimed to explore the toxicological impacts of microinjection of remdesivir on zebrafish early embryonic development and larvae and the associated mechanism. We found that 100 μM remdesivir delayed epiboly and impaired convergent movement of embryos during gastrulation, and dose-dependent increases in mortality and malformation were observed in remdesivir-treated embryos. Moreover, 10-100 μM remdesivir decreased blood flow and swimming velocity and altered the behavior of larvae. In terms of molecular mechanisms, eighty differentially expressed genes (DEGs) were identified by transcriptome analysis in the remdesivir-treated group. Some of these DEGs, such as manf, kif3a, hnf1ba, rgn, prkcz, egr1, fosab, nr4a1, and ptgs2b, were mainly involved in early embryonic development, neuronal developmental disorders, vascular disease and the blood flow pathway. These data reveal that remdesivir can impair early embryonic development, blood flow and behavior of zebrafish embryos/larvae, probably due to alterations at the transcriptome level. This study suggests that it is important to avoid the discharge of remdesivir to aquatic ecosystems and provides a theoretical foundation to hinder remdesivir-induced ecotoxicity to aquatic environments.
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Affiliation(s)
- Ji-Tong Li
- Henan Neurodevelopment Engineering Research Center for Children; Henan Provincial Key Laboratory of Children's Genetics and Metabolic Diseases, Children's Hospital Affiliated to Zhengzhou University, Henan Children's Hospital, Zhengzhou Children's Hospital, Zhengzhou, China.,Department of Nephrology and Rheumatology, Children's Hospital Affiliated to Zhengzhou University; Zhengzhou Key Laboratory of Pediatric Kidney Disease Research, Zhengzhou, China
| | - Yao-Dong Zhang
- Henan Neurodevelopment Engineering Research Center for Children; Henan Provincial Key Laboratory of Children's Genetics and Metabolic Diseases, Children's Hospital Affiliated to Zhengzhou University, Henan Children's Hospital, Zhengzhou Children's Hospital, Zhengzhou, China
| | - Xiao-Rui Song
- Henan Neurodevelopment Engineering Research Center for Children; Henan Provincial Key Laboratory of Children's Genetics and Metabolic Diseases, Children's Hospital Affiliated to Zhengzhou University, Henan Children's Hospital, Zhengzhou Children's Hospital, Zhengzhou, China
| | - Rui-Jing Li
- Henan Neurodevelopment Engineering Research Center for Children; Henan Provincial Key Laboratory of Children's Genetics and Metabolic Diseases, Children's Hospital Affiliated to Zhengzhou University, Henan Children's Hospital, Zhengzhou Children's Hospital, Zhengzhou, China
| | - Wei-Li Yang
- Henan Neurodevelopment Engineering Research Center for Children; Henan Provincial Key Laboratory of Children's Genetics and Metabolic Diseases, Children's Hospital Affiliated to Zhengzhou University, Henan Children's Hospital, Zhengzhou Children's Hospital, Zhengzhou, China
| | - Ming Tian
- Department of Nephrology and Rheumatology, Children's Hospital Affiliated to Zhengzhou University; Zhengzhou Key Laboratory of Pediatric Kidney Disease Research, Zhengzhou, China
| | - Shu-Feng Zhang
- Department of Nephrology and Rheumatology, Children's Hospital Affiliated to Zhengzhou University; Zhengzhou Key Laboratory of Pediatric Kidney Disease Research, Zhengzhou, China
| | - Guang-Hai Cao
- Department of Nephrology and Rheumatology, Children's Hospital Affiliated to Zhengzhou University; Zhengzhou Key Laboratory of Pediatric Kidney Disease Research, Zhengzhou, China
| | - Lu-Lu Song
- School of Public Health, Zhengzhou University, Zhengzhou, China
| | - Yu-Ming Chen
- School of Public Health, Xinxiang Medical University, Xinxiang, China
| | - Cui-Hua Liu
- Henan Neurodevelopment Engineering Research Center for Children; Henan Provincial Key Laboratory of Children's Genetics and Metabolic Diseases, Children's Hospital Affiliated to Zhengzhou University, Henan Children's Hospital, Zhengzhou Children's Hospital, Zhengzhou, China.,Department of Nephrology and Rheumatology, Children's Hospital Affiliated to Zhengzhou University; Zhengzhou Key Laboratory of Pediatric Kidney Disease Research, Zhengzhou, China
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20
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Visualizing multiciliated cells in the zebrafish. Methods Cell Biol 2022. [DOI: 10.1016/bs.mcb.2022.09.008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
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21
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Klingbeil K, Nguyen TQ, Fahrner A, Guthmann C, Wang H, Schoels M, Lilienkamp M, Franz H, Eckert P, Walz G, Yakulov TA. Corpuscles of Stannius development requires FGF signaling. Dev Biol 2021; 481:160-171. [PMID: 34666023 DOI: 10.1016/j.ydbio.2021.10.005] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2021] [Revised: 09/06/2021] [Accepted: 10/11/2021] [Indexed: 01/02/2023]
Abstract
The corpuscles of Stannius (CS) represent a unique endocrine organ of teleostean fish that secrets stanniocalcin-1 (Stc1) to maintain calcium homeostasis. Appearing at 20-25 somite stage in the distal zebrafish pronephros, stc1-expressing cells undergo apical constriction, and are subsequently extruded to form a distinct gland on top of the distal pronephric tubules at 50 h post fertilization (hpf). Several transcription factors (e.g. Hnf1b, Irx3b, Tbx2a/b) and signaling pathways (e.g. Notch) control CS development. We report now that Fgf signaling is required to commit tubular epithelial cells to differentiate into stc1-expressing CS cells. Inhibition of Fgf signaling by SU5402, dominant-negative Fgfr1, or depletion of fgf8a prevented CS formation and stc1 expression. Ablation experiments revealed that CS have the ability to partially regenerate via active cell migration involving extensive filopodia and lamellipodia formation. Activation of Wnt signaling curtailed stc1 expression, but had no effect on CS formation. Thus, our observations identify Fgf signaling as a crucial component of CS cell fate commitment.
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Affiliation(s)
- Konstantin Klingbeil
- Renal Division, Department of Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany
| | - Thanh Quang Nguyen
- Renal Division, Department of Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany
| | - Andreas Fahrner
- Renal Division, Department of Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany
| | - Clara Guthmann
- Renal Division, Department of Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany
| | - Hui Wang
- Renal Division, Department of Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany
| | - Maximilian Schoels
- Renal Division, Department of Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany
| | - Miriam Lilienkamp
- Renal Division, Department of Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany
| | - Henriette Franz
- Department of Biomedicine, University of Basel, Basel, Switzerland
| | - Priska Eckert
- Renal Division, Department of Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany
| | - Gerd Walz
- Renal Division, Department of Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany; Signaling Research Centers BIOSS and CIBSS, University of Freiburg, Albertstrasse 19, 79104, Freiburg, Germany
| | - Toma Antonov Yakulov
- Renal Division, Department of Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany.
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22
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Piedrafita A, Balayssac S, Casemayou A, Saulnier-Blache JS, Lucas A, Iacovoni JS, Breuil B, Chauveau D, Decramer S, Malet-Martino M, Schanstra JP, Faguer S. Hepatocyte nuclear factor-1β shapes the energetic homeostasis of kidney tubule cells. FASEB J 2021; 35:e21931. [PMID: 34653285 DOI: 10.1096/fj.202100782rr] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2021] [Revised: 08/28/2021] [Accepted: 09/02/2021] [Indexed: 12/17/2022]
Abstract
Energetic metabolism controls key steps of kidney development, homeostasis, and epithelial repair following acute kidney injury (AKI). Hepatocyte nuclear factor-1β (HNF-1β) is a master transcription factor that controls mitochondrial function in proximal tubule (PT) cells. Patients with HNF1B pathogenic variant display a wide range of kidney developmental abnormalities and progressive kidney fibrosis. Characterizing the metabolic changes in PT cells with HNF-1β deficiency may help to identify new targetable molecular hubs involved in HNF1B-related kidney phenotypes and AKI. Here, we combined 1 H-NMR-based metabolomic analysis in a murine PT cell line with CrispR/Cas9-induced Hnf1b invalidation (Hnf1b-/- ), clustering analysis, targeted metabolic assays, and datamining of published RNA-seq and ChIP-seq dataset to identify the role of HNF-1β in metabolism. Hnf1b-/- cells grown in normoxic conditions display intracellular ATP depletion, increased cytosolic lactate concentration, increased lipid droplet content, failure to use pyruvate for energetic purposes, increased levels of tricarboxylic acid (TCA) cycle intermediates and oxidized glutathione, and a reduction of TCA cycle byproducts, all features consistent with mitochondrial dysfunction and an irreversible switch toward glycolysis. Unsupervised clustering analysis showed that Hnf1b-/- cells mimic a hypoxic signature and that they cannot furthermore increase glycolysis-dependent energetic supply during hypoxic challenge. Metabolome analysis also showed alteration of phospholipid biosynthesis in Hnf1b-/- cells leading to the identification of Chka, the gene coding for choline kinase α, as a new putative target of HNF-1β. HNF-1β shapes the energetic metabolism of PT cells and HNF1B deficiency in patients could lead to a hypoxia-like metabolic state precluding further adaptation to ATP depletion following AKI.
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Affiliation(s)
- Alexis Piedrafita
- Institut National de la Santé et de la Recherche Médicale, UMR 1297, Institut des Maladies Métaboliques et Cardiovasculaires, Hôpital Rangueil, Toulouse, France.,Université Paul Sabatier - Toulouse 3, Toulouse, France.,Département de Néphrologie et Transplantation d'Organes, Centre de Référence des Maladies Rénales Rares, Centre Hospitalier Universitaire de Toulouse, Toulouse, France
| | - Stéphane Balayssac
- Groupe de RMN Biomédicale, Laboratoire SPCMIB, UMR CNRS 5068, Université Paul Sabatier, Centre National de la Recherche Scientifique, Toulouse, France.,Laboratoire des Interaction Moléculaires et Réactivité Chimique et Photochimique (IMRCP), UMR 5623, Toulouse, France
| | - Audrey Casemayou
- Institut National de la Santé et de la Recherche Médicale, UMR 1297, Institut des Maladies Métaboliques et Cardiovasculaires, Hôpital Rangueil, Toulouse, France.,Université Paul Sabatier - Toulouse 3, Toulouse, France.,Département de Néphrologie et Transplantation d'Organes, Centre de Référence des Maladies Rénales Rares, Centre Hospitalier Universitaire de Toulouse, Toulouse, France
| | - Jean-Sébastien Saulnier-Blache
- Institut National de la Santé et de la Recherche Médicale, UMR 1297, Institut des Maladies Métaboliques et Cardiovasculaires, Hôpital Rangueil, Toulouse, France.,Université Paul Sabatier - Toulouse 3, Toulouse, France
| | - Alexandre Lucas
- Institut National de la Santé et de la Recherche Médicale, UMR 1297, Institut des Maladies Métaboliques et Cardiovasculaires, Hôpital Rangueil, Toulouse, France
| | - Jason S Iacovoni
- Institut National de la Santé et de la Recherche Médicale, UMR 1297, Institut des Maladies Métaboliques et Cardiovasculaires, Hôpital Rangueil, Toulouse, France
| | - Benjamin Breuil
- Institut National de la Santé et de la Recherche Médicale, UMR 1297, Institut des Maladies Métaboliques et Cardiovasculaires, Hôpital Rangueil, Toulouse, France
| | - Dominique Chauveau
- Institut National de la Santé et de la Recherche Médicale, UMR 1297, Institut des Maladies Métaboliques et Cardiovasculaires, Hôpital Rangueil, Toulouse, France.,Université Paul Sabatier - Toulouse 3, Toulouse, France.,Département de Néphrologie et Transplantation d'Organes, Centre de Référence des Maladies Rénales Rares, Centre Hospitalier Universitaire de Toulouse, Toulouse, France
| | - Stéphane Decramer
- Institut National de la Santé et de la Recherche Médicale, UMR 1297, Institut des Maladies Métaboliques et Cardiovasculaires, Hôpital Rangueil, Toulouse, France.,Université Paul Sabatier - Toulouse 3, Toulouse, France.,Service de Néphrologie, Médecine interne et Hypertension artérielle, Hôpital des Enfants, Centre de Référence des Maladies Rénales Rares, Centre Hospitalier Universitaire de Toulouse, Toulouse, France
| | - Myriam Malet-Martino
- Groupe de RMN Biomédicale, Laboratoire SPCMIB, UMR CNRS 5068, Université Paul Sabatier, Centre National de la Recherche Scientifique, Toulouse, France
| | - Joost P Schanstra
- Institut National de la Santé et de la Recherche Médicale, UMR 1297, Institut des Maladies Métaboliques et Cardiovasculaires, Hôpital Rangueil, Toulouse, France.,Université Paul Sabatier - Toulouse 3, Toulouse, France
| | - Stanislas Faguer
- Institut National de la Santé et de la Recherche Médicale, UMR 1297, Institut des Maladies Métaboliques et Cardiovasculaires, Hôpital Rangueil, Toulouse, France.,Université Paul Sabatier - Toulouse 3, Toulouse, France.,Département de Néphrologie et Transplantation d'Organes, Centre de Référence des Maladies Rénales Rares, Centre Hospitalier Universitaire de Toulouse, Toulouse, France
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23
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Pou Casellas C, Jansen K, Rookmaaker MB, Clevers H, Verhaar MC, Masereeuw R. Regulation of Solute Carriers OCT2 and OAT1/3 in the Kidney: A Phylogenetic, Ontogenetic and Cell Dynamic Perspective. Physiol Rev 2021; 102:993-1024. [PMID: 34486394 DOI: 10.1152/physrev.00009.2021] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Over the course of more than 500 million years, the kidneys have undergone a remarkable evolution from primitive nephric tubes to intricate filtration-reabsorption systems that maintain homeostasis and remove metabolic end products from the body. The evolutionarily conserved solute carriers Organic Cation Transporter 2 (OCT2), and Organic Anion Transporters 1 and 3 (OAT1/3) coordinate the active secretion of a broad range of endogenous and exogenous substances, many of which accumulate in the blood of patients with kidney failure despite dialysis. Harnessing OCT2 and OAT1/3 through functional preservation or regeneration could alleviate the progression of kidney disease. Additionally, it would improve current in vitro test models that lose their expression in culture. With this review, we explore OCT2 and OAT1/3 regulation using different perspectives: phylogenetic, ontogenetic and cell dynamic. Our aim is to identify possible molecular targets to both help prevent or compensate for the loss of transport activity in patients with kidney disease, and to enable endogenous OCT2 and OAT1/3 induction in vitro in order to develop better models for drug development.
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Affiliation(s)
- Carla Pou Casellas
- Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, Netherlands.,Hubrecht Institute - Royal Netherlands Academy of Arts and Sciences, Utrecht, The Netherlands
| | - Katja Jansen
- Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands
| | - Maarten B Rookmaaker
- Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, Netherlands
| | - Hans Clevers
- Hubrecht Institute - Royal Netherlands Academy of Arts and Sciences, Utrecht, The Netherlands
| | - Marianne C Verhaar
- Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, Netherlands
| | - Rosalinde Masereeuw
- Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands
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24
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Chambers JM, Wingert RA. Advances in understanding vertebrate nephrogenesis. Tissue Barriers 2020; 8:1832844. [PMID: 33092489 PMCID: PMC7714473 DOI: 10.1080/21688370.2020.1832844] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2020] [Revised: 10/01/2020] [Accepted: 10/01/2020] [Indexed: 02/07/2023] Open
Abstract
The kidney is a complex organ that performs essential functions such as blood filtration and fluid homeostasis, among others. Recent years have heralded significant advancements in our knowledge of the mechanisms that control kidney formation. Here, we provide an overview of vertebrate renal development with a focus on nephrogenesis, the process of generating the epithelialized functional units of the kidney. These steps begin with intermediate mesoderm specification and proceed all the way to the terminally differentiated nephron cell, with many detailed stages in between. The establishment of nephron architecture with proper cellular barriers is vital throughout these processes. Continuously striving to gain further insights into nephrogenesis can ultimately lead to a better understanding and potential treatments for developmental maladies such as Congenital Anomalies of the Kidney and Urinary Tract (CAKUT).
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Affiliation(s)
- Joseph M. Chambers
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, Boler-Parseghian Center for Rare and Neglected Diseases, University of Notre Dame, Notre Dame, IN, USA
| | - Rebecca A. Wingert
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, Boler-Parseghian Center for Rare and Neglected Diseases, University of Notre Dame, Notre Dame, IN, USA
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25
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Preya UH, Woo JH, Choi YS, Choi JH. Hepatocyte nuclear factor-1 beta protects endometriotic cells against apoptotic cell death by up-regulating the expression of antiapoptotic genes†. Biol Reprod 2019; 101:686-694. [PMID: 31322170 DOI: 10.1093/biolre/ioz127] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2019] [Revised: 06/07/2019] [Accepted: 07/11/2019] [Indexed: 12/17/2022] Open
Abstract
The overexpression of hepatocyte nuclear factor-1 beta (HNF1β) in endometriotic lesion has been demonstrated. However, the role of HNF1β in endometriosis remains largely unknown. Human endometriotic 12Z cells showed higher level of HNF1β when compared with normal endometrial HES cells. In human endometriotic 12Z cells, HNF1β knockdown increased susceptibility to apoptotic cell death by oxidative stress, while HNF1β overexpression suppressed apoptosis. In addition, HNF1β knockdown and overexpression significantly decreased and increased, respectively, the expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-dependent antiapoptotic genes. Knockdown of the antiapoptotic genes significantly reduced the HNF1β-induced resistance against oxidative stress in 12Z cells. Furthermore, HNF1β regulated the transcriptional activity of NF-κB, and an NF-κB inhibitor suppressed the HNF1β-enhanced NF-κB-dependent antiapoptotic gene expression and the resistance of the 12Z cells against cell death. Taken together, these data suggest that HNF1β overexpression may protect endometriotic cells against oxidative damage by augmenting antiapoptotic gene expression.
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Affiliation(s)
- Umma Hafsa Preya
- College of Pharmacy, Kyung Hee University, Seoul, South Korea.,Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul, South Korea
| | - Jeong-Hwa Woo
- College of Pharmacy, Kyung Hee University, Seoul, South Korea
| | - Youn Seok Choi
- Department of Obstetrics and Gynecology, School of Medicine, Catholic University of Daegu, Daegu, South Korea
| | - Jung-Hye Choi
- College of Pharmacy, Kyung Hee University, Seoul, South Korea.,Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul, South Korea
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26
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Ferrè S, Igarashi P. New insights into the role of HNF-1β in kidney (patho)physiology. Pediatr Nephrol 2019; 34:1325-1335. [PMID: 29961928 PMCID: PMC6312759 DOI: 10.1007/s00467-018-3990-7] [Citation(s) in RCA: 59] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/30/2018] [Revised: 05/24/2018] [Accepted: 05/25/2018] [Indexed: 12/14/2022]
Abstract
Hepatocyte nuclear factor-1β (HNF-1β) is an essential transcription factor that regulates the development and function of epithelia in the kidney, liver, pancreas, and genitourinary tract. Humans who carry HNF1B mutations develop heterogeneous renal abnormalities, including multicystic dysplastic kidneys, glomerulocystic kidney disease, renal agenesis, renal hypoplasia, and renal interstitial fibrosis. In the embryonic kidney, HNF-1β is required for ureteric bud branching, initiation of nephrogenesis, and nephron segmentation. Ablation of mouse Hnf1b in nephron progenitors causes defective tubulogenesis, whereas later inactivation in elongating tubules leads to cyst formation due to downregulation of cystic disease genes, including Umod, Pkhd1, and Pkd2. In the adult kidney, HNF-1β controls the expression of genes required for intrarenal metabolism and solute transport by tubular epithelial cells. Tubular abnormalities observed in HNF-1β nephropathy include hyperuricemia with or without gout, hypokalemia, hypomagnesemia, and polyuria. Recent studies have identified novel post-transcriptional and post-translational regulatory mechanisms that control HNF-1β expression and activity, including the miRNA cluster miR17 ∼ 92 and the interacting proteins PCBD1 and zyxin. Further understanding of the molecular mechanisms upstream and downstream of HNF-1β may lead to the development of new therapeutic approaches in cystic kidney disease and other HNF1B-related renal diseases.
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Affiliation(s)
- Silvia Ferrè
- Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, Texas, USA,Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Peter Igarashi
- Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA. .,Department of Medicine, University of Minnesota Medical School, 420 Delaware St. SE, MMC 194, Minneapolis, MN, 55455, USA.
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27
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Sander V, Salleh L, Naylor RW, Schierding W, Sontam D, O’Sullivan JM, Davidson AJ. Transcriptional profiling of the zebrafish proximal tubule. Am J Physiol Renal Physiol 2019; 317:F478-F488. [DOI: 10.1152/ajprenal.00174.2019] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
The hepatocyte nuclear factor-1β (Hnf1b) transcription factor is a key regulator of kidney tubule formation and is associated with a syndrome of renal cysts and early onset diabetes. To further our understanding of Hnf1b in the developing zebrafish kidney, we performed RNA sequencing analysis of proximal tubules from hnf1b-deficient larvae. This analysis revealed an enrichment of gene transcripts encoding transporters of the solute carrier (SLC) superfamily, including multiple members of slc2 and slc5 glucose transporters. An investigation of expression of slc2a1a, slc2a2, and slc5a2 as well as a poorly studied glucose/mannose transporter encoded by slc5a9 revealed that these genes undergo dynamic spatiotemporal changes during tubule formation and maturation. A comparative analysis of zebrafish SLC genes with those expressed in mouse proximal tubules showed a substantial overlap at the level of gene families, indicating a high degree of functional conservation between zebrafish and mammalian proximal tubules. Taken together, our findings are consistent with a role for Hnf1b as a critical determinant of proximal tubule transport function by acting upstream of a large number of SLC genes and validate the zebrafish as a physiologically relevant model of the mammalian proximal tubule.
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Affiliation(s)
- Veronika Sander
- Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand
| | - Liam Salleh
- Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand
| | - Richard W. Naylor
- Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand
| | | | - Dharani Sontam
- The Liggins Institute, University of Auckland, Auckland, New Zealand
| | | | - Alan J. Davidson
- Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand
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28
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Abstract
The vertebrate kidney is comprised of functional units known as nephrons. Defects in nephron development or activity are a common feature of kidney disease. Current medical treatments are unable to ameliorate the dire consequences of nephron deficit or injury. Although there have been tremendous advancements in our understanding of nephron ontogeny and the response to damage, many significant knowledge gaps still remain. The zebrafish embryo kidney, or pronephros, is an ideal model for many renal development and regeneration studies because it is comprised of nephrons that share conserved features with the nephron units that comprise the mammalian metanephric kidney. In this chapter, we provide an overview about the benefits of using the zebrafish pronephros to study the mechanisms underlying nephrogenesis as well as epithelial repair and regeneration. We subsequently detail methods for the spatiotemporal assessment of gene and protein expression in zebrafish embryos that can be used to extend the understanding of nephron development and disease, and thereby create new opportunities to identify therapeutic strategies for regenerative medicine.
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29
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Chambers BE, Gerlach GF, Clark EG, Chen KH, Levesque AE, Leshchiner I, Goessling W, Wingert RA. Tfap2a is a novel gatekeeper of nephron differentiation during kidney development. Development 2019; 146:dev.172387. [PMID: 31160420 DOI: 10.1242/dev.172387] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2018] [Accepted: 05/22/2019] [Indexed: 12/13/2022]
Abstract
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.
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Affiliation(s)
- Brooke E Chambers
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Gary F Gerlach
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Eleanor G Clark
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Karen H Chen
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Anna E Levesque
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Ignaty Leshchiner
- Brigham and Women's Hospital, Genetics and Gastroenterology Division, Harvard Medical School, Harvard Stem Cell Institute, Boston, MA 02215, USA
| | - Wolfram Goessling
- Brigham and Women's Hospital, Genetics and Gastroenterology Division, Harvard Medical School, Harvard Stem Cell Institute, Boston, MA 02215, USA
| | - Rebecca A Wingert
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
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30
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Chambers BE, Wingert RA. Mechanisms of Nephrogenesis Revealed by Zebrafish Chemical Screen: Prostaglandin Signaling Modulates Nephron Progenitor Fate. Nephron Clin Pract 2019; 143:68-76. [PMID: 31216548 DOI: 10.1159/000501037] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2018] [Accepted: 05/17/2019] [Indexed: 12/15/2022] Open
Abstract
Nephron development involves the creation of discrete segment populations that are specialized to fulfill unique physiological roles. As such, renal function is reliant on the proper execution of segment patterning programs. Despite the central importance of nephron segmentation, the genetic mechanisms that regulate this process are far from understood, in large part due to the experimental complexities and cost of interrogating these events in the mammalian metanephros. For this reason, forward genetics utilizing phenotypic screening in the zebrafish pronephros provides an avenue to gain novel insights about the mechanisms of nephron segmentation in the vertebrate kidney. Discoveries from zebrafish can highlight possible conserved pathways and provide a useful starting point for reverse genetic analyses with other animal models or in vitro approaches. In this review, we discuss the results of a novel chemical screen using the zebrafish to identify segmentation regulators. Through this screen, we identified for the first time that prostaglandin signaling can modulate nephron segmentation, and that it is normally requisite during development to mitigate segment fate choice in the embryonic kidney. We briefly discuss how these discoveries relate to current knowledge about nephron segmentation. Finally, we explore the possible implications of these findings for understanding renal ontogeny and disease, and how this knowledge may be useful for ongoing research initiatives that are aimed at deciphering how to build or rebuild the human kidney.
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Affiliation(s)
- Brooke E Chambers
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, Indiana, USA
| | - Rebecca A Wingert
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, Indiana, USA,
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31
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Adalat S, Hayes WN, Bryant WA, Booth J, Woolf AS, Kleta R, Subtil S, Clissold R, Colclough K, Ellard S, Bockenhauer D. HNF1B Mutations Are Associated With a Gitelman-like Tubulopathy That Develops During Childhood. Kidney Int Rep 2019; 4:1304-1311. [PMID: 31517149 PMCID: PMC6732753 DOI: 10.1016/j.ekir.2019.05.019] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2019] [Revised: 04/29/2019] [Accepted: 05/20/2019] [Indexed: 12/20/2022] Open
Abstract
Background Mutations in the transcription factor hepatocyte nuclear factor 1B (HNF1B) are the most common inherited cause of renal malformations, yet also associated with renal tubular dysfunction, most prominently magnesium wasting with hypomagnesemia. The presence of hypomagnesemia has been proposed to help select appropriate patients for genetic testing. Yet, in a large cohort, hypomagnesemia was discriminatory only in adult, but not in pediatric patients. We therefore investigated whether hypomagnesemia and other biochemical changes develop with age. Methods We performed a retrospective analysis of clinical, biochemical, and genetic results of pediatric patients with renal malformations tested for HNF1B mutations, separated into 4 age groups. Values were excluded if concurrent estimated glomerular filtration rate (eGFR) was <30 ml/min per 1.73 m2, or after transplantation. Results A total of 199 patients underwent HNF1B genetic testing and mutations were identified in 52 (mut+). The eGFRs were comparable between mut+ and mut- in any age group. Although median plasma magnesium concentrations differed significantly between mut+ and mut- patients in all age groups, overt hypomagnesemia was not present until the second half of childhood in the mut+ group. There was also a significant difference in median potassium concentrations in late childhood with lower values in the mut+ cohort. Conclusions The abnormal tubular electrolyte handling associated with HNF1B mutations develops with age and is not restricted to magnesium, but consistent with a more generalized dysfunction of the distal convoluted tubule, reminiscent of Gitelman syndrome. The absence of these abnormalities in early childhood should not preclude HNF1B mutations from diagnostic considerations.
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Affiliation(s)
- Shazia Adalat
- Evelina Children’s Hospital, London, United Kingdom
- UCL Department of Renal Medicine, London, United Kingdom
| | - Wesley N. Hayes
- UCL Department of Renal Medicine, London, United Kingdom
- Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom
| | - William A. Bryant
- Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom
| | - John Booth
- Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom
| | - Adrian S. Woolf
- Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology Medicine and Health, University of Manchester, United Kingdom
- Royal Manchester Children’s Hospital, Manchester University NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - Robert Kleta
- UCL Department of Renal Medicine, London, United Kingdom
- Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom
| | | | - Rhian Clissold
- Royal Devon and Exeter NHS Foundation Trust, Exeter, United Kingdom
| | - Kevin Colclough
- Department of Molecular Genetics, Royal Devon & Exeter NHS Foundation Trust, Exeter, United Kingdom
| | - Sian Ellard
- Department of Molecular Genetics, Royal Devon & Exeter NHS Foundation Trust, Exeter, United Kingdom
- Institute of Biomedical and Clinical Science, College of Medicine and Health, University of Exeter, Exeter, United Kingdom
| | - Detlef Bockenhauer
- UCL Department of Renal Medicine, London, United Kingdom
- Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom
- Correspondence: Detlef Bockenhauer, UCL Department of Renal Medicine, London WC1N 3JH, United Kingdom.
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32
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Naylor RW, Chang HHG, Qubisi S, Davidson AJ. A novel mechanism of gland formation in zebrafish involving transdifferentiation of renal epithelial cells and live cell extrusion. eLife 2018; 7:38911. [PMID: 30394875 PMCID: PMC6250424 DOI: 10.7554/elife.38911] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2018] [Accepted: 11/05/2018] [Indexed: 12/12/2022] Open
Abstract
Transdifferentiation is the poorly understood phenomenon whereby a terminally differentiated cell acquires a completely new identity. Here, we describe a rare example of a naturally occurring transdifferentiation event in zebrafish in which kidney distal tubule epithelial cells are converted into an endocrine gland known as the Corpuscles of Stannius (CS). We find that this process requires Notch signalling and is associated with the cytoplasmic sequestration of the Hnf1b transcription factor, a master-regulator of renal tubule fate. A deficiency in the Irx3b transcription factor results in ectopic transdifferentiation of distal tubule cells to a CS identity but in a Notch-dependent fashion. Using live-cell imaging we show that CS cells undergo apical constriction en masse and are then extruded from the tubule to form a distinct organ. This system provides a valuable new model to understand the molecular and morphological basis of transdifferentiation and will advance efforts to exploit this rare phenomenon therapeutically.
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Affiliation(s)
- Richard W Naylor
- Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand
| | - Hao-Han G Chang
- Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand
| | - Sarah Qubisi
- Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand
| | - Alan J Davidson
- Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand
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33
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Barrodia P, Patra C, Swain RK. EF-hand domain containing 2 (Efhc2) is crucial for distal segmentation of pronephros in zebrafish. Cell Biosci 2018; 8:53. [PMID: 30349665 PMCID: PMC6192171 DOI: 10.1186/s13578-018-0253-z] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2018] [Accepted: 10/05/2018] [Indexed: 12/31/2022] Open
Abstract
Background The blood filtering organ in zebrafish embryos is the pronephros, which consists of two functional nephrons. Segmentation of a nephron into different domains is essential for its function and is well conserved among vertebrates. Zebrafish has been extensively used as a model to understand nephron segmentation during development. Here, we have identified EF-hand domain containing 2 (Efhc2) as a novel component of genetic programme regulating nephron segmentation in zebrafish. Human EFHC2 is a protein with one predicted calcium-binding EF-hand motif and three DM10 domains, whose function is unknown. EFHC2 has been implicated in several brain-related genetic diseases like Turner syndrome and juvenile myoclonic epilepsy. However, there is limited information on its normal physiological function. Results efhc2 mRNA is primarily expressed in the pronephros of zebrafish embryos. Other sites of expression include olfactory placode, notochord, otic vesicle, epiphysis and neuromast cells. Morpholino antisense oligonucleotide-mediated knock-down of Efhc2 resulted in defects in pronephros development and function in zebrafish embryos. Efhc2 knock-down leads to expansion of distal early segment of pronephros, whereas, the corpuscle of stannius and distal late segments were reduced. The number of multi-ciliated cells (MCC) that are present in a salt-and-pepper fashion throughout the middle of each nephron and vital for fluid flow were also reduced. It is known that retinoic acid (RA) signaling regulates pronephros segmentation in vertebrates and we show that Efhc2 function is crucial for nephron segmentation in zebrafish. Our data suggests that RA and Efhc2 function independent of each other in pronephros segmentation. However, Efhc2 and RA synergistically regulate MCC development. Conclusion In this study, we have identified Efhc2 as a regulator of segmentation of the distal part of nephron and pronephros function during zebrafish development. Electronic supplementary material The online version of this article (10.1186/s13578-018-0253-z) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Praveen Barrodia
- 1Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, Odisha 751023 India.,2Manipal Academy of Higher Education, Manipal, Karnataka 576104 India
| | - Chinmoy Patra
- 3Agharkar Research Institute, Pune, Maharashtra India.,4Savitribai Phule Pune University, Pune, Maharashtra India
| | - Rajeeb K Swain
- 1Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, Odisha 751023 India
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Elmonem MA, Berlingerio SP, van den Heuvel LP, de Witte PA, Lowe M, Levtchenko EN. Genetic Renal Diseases: The Emerging Role of Zebrafish Models. Cells 2018; 7:cells7090130. [PMID: 30200518 PMCID: PMC6162634 DOI: 10.3390/cells7090130] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2018] [Revised: 08/27/2018] [Accepted: 08/29/2018] [Indexed: 12/14/2022] Open
Abstract
The structural and functional similarity of the larval zebrafish pronephros to the human nephron, together with the recent development of easier and more precise techniques to manipulate the zebrafish genome have motivated many researchers to model human renal diseases in the zebrafish. Over the last few years, great advances have been made, not only in the modeling techniques of genetic diseases in the zebrafish, but also in how to validate and exploit these models, crossing the bridge towards more informative explanations of disease pathophysiology and better designed therapeutic interventions in a cost-effective in vivo system. Here, we review the significant progress in these areas giving special attention to the renal phenotype evaluation techniques. We further discuss the future applications of such models, particularly their role in revealing new genetic diseases of the kidney and their potential use in personalized medicine.
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Affiliation(s)
- Mohamed A Elmonem
- Department of Pediatric Nephrology & Development and Regeneration, University Hospitals Leuven, KU Leuven-University of Leuven, Herestraat 49, Box 817, 3000 Leuven, Belgium.
- Department of Clinical and Chemical Pathology, Faculty of Medicine, Cairo University, 11628 Cairo, Egypt.
| | - Sante Princiero Berlingerio
- Department of Pediatric Nephrology & Development and Regeneration, University Hospitals Leuven, KU Leuven-University of Leuven, Herestraat 49, Box 817, 3000 Leuven, Belgium.
| | - Lambertus P van den Heuvel
- Department of Pediatric Nephrology & Development and Regeneration, University Hospitals Leuven, KU Leuven-University of Leuven, Herestraat 49, Box 817, 3000 Leuven, Belgium.
- Department of Pediatric Nephrology, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.
| | - Peter A de Witte
- Laboratory for Molecular Bio-Discovery, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven-University of Leuven, 3000 Leuven, Belgium.
| | - Martin Lowe
- Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PL, UK.
| | - Elena N Levtchenko
- Department of Pediatric Nephrology & Development and Regeneration, University Hospitals Leuven, KU Leuven-University of Leuven, Herestraat 49, Box 817, 3000 Leuven, Belgium.
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Disruption of mesoderm formation during cardiac differentiation due to developmental exposure to 13-cis-retinoic acid. Sci Rep 2018; 8:12960. [PMID: 30154523 PMCID: PMC6113333 DOI: 10.1038/s41598-018-31192-0] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2018] [Accepted: 08/14/2018] [Indexed: 12/18/2022] Open
Abstract
13-cis-retinoic acid (isotretinoin, INN) is an oral pharmaceutical drug used for the treatment of skin acne, and is also a known teratogen. In this study, the molecular mechanisms underlying INN-induced developmental toxicity during early cardiac differentiation were investigated using both human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). Pre-exposure of hiPSCs and hESCs to a sublethal concentration of INN did not influence cell proliferation and pluripotency. However, mesodermal differentiation was disrupted when INN was included in the medium during differentiation. Transcriptomic profiling by RNA-seq revealed that INN exposure leads to aberrant expression of genes involved in several signaling pathways that control early mesoderm differentiation, such as TGF-beta signaling. In addition, genome-wide chromatin accessibility profiling by ATAC-seq suggested that INN-exposure leads to enhanced DNA-binding of specific transcription factors (TFs), including HNF1B, SOX10 and NFIC, often in close spatial proximity to genes that are dysregulated in response to INN treatment. Altogether, these results identify potential molecular mechanisms underlying INN-induced perturbation during mesodermal differentiation in the context of cardiac development. This study further highlights the utility of human stem cells as an alternative system for investigating congenital diseases of newborns that arise as a result of maternal drug exposure during pregnancy.
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Przepiorski A, Sander V, Tran T, Hollywood JA, Sorrenson B, Shih JH, Wolvetang EJ, McMahon AP, Holm TM, Davidson AJ. A Simple Bioreactor-Based Method to Generate Kidney Organoids from Pluripotent Stem Cells. Stem Cell Reports 2018; 11:470-484. [PMID: 30033089 PMCID: PMC6092837 DOI: 10.1016/j.stemcr.2018.06.018] [Citation(s) in RCA: 174] [Impact Index Per Article: 24.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2017] [Revised: 06/21/2018] [Accepted: 06/22/2018] [Indexed: 02/07/2023] Open
Abstract
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.
Technically simple and cost-efficient protocol for kidney organoid generation Tubular organoids are obtained rapidly, with high efficiency, yield, and robustness Organoids contain nephrons that correspond to human fetal nephrons The applicability to model congenital kidney defects is presented
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Affiliation(s)
- Aneta Przepiorski
- Department of Molecular Medicine & Pathology, University of Auckland, Auckland 1142, New Zealand
| | - Veronika Sander
- Department of Molecular Medicine & Pathology, University of Auckland, Auckland 1142, New Zealand
| | - Tracy Tran
- Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Jennifer A Hollywood
- Department of Molecular Medicine & Pathology, University of Auckland, Auckland 1142, New Zealand
| | - Brie Sorrenson
- Department of Molecular Medicine & Pathology, University of Auckland, Auckland 1142, New Zealand
| | - Jen-Hsing Shih
- Department of Molecular Medicine & Pathology, University of Auckland, Auckland 1142, New Zealand
| | - Ernst J Wolvetang
- Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, QLD 4072, Australia
| | - Andrew P McMahon
- Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Teresa M Holm
- Department of Molecular Medicine & Pathology, University of Auckland, Auckland 1142, New Zealand
| | - Alan J Davidson
- Department of Molecular Medicine & Pathology, University of Auckland, Auckland 1142, New Zealand.
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Xu D, Lv J, He L, Fu L, Hu R, Cao Y, Mei C. Scribble influences cyst formation in autosomal‐dominant polycystic kidney disease by regulating Hippo signaling pathway. FASEB J 2018. [DOI: 10.1096/fj.201701376rr] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Affiliation(s)
- Dechao Xu
- Department of NephrologyKidney Institute of the People's Liberation ArmyChangzheng HospitalSecond Military Medical UniversityShanghaiChina
| | - Jiayi Lv
- Department of NephrologyKidney Institute of the People's Liberation ArmyChangzheng HospitalSecond Military Medical UniversityShanghaiChina
| | - Liangliang He
- Department of NephrologyKidney Institute of the People's Liberation ArmyChangzheng HospitalSecond Military Medical UniversityShanghaiChina
| | - Lili Fu
- Department of NephrologyKidney Institute of the People's Liberation ArmyChangzheng HospitalSecond Military Medical UniversityShanghaiChina
| | - Ruikun Hu
- Department of Molecular and Cell BiologySchool of Life Sciences and TechnologyTongi UniversityShanghaiChina
| | - Ying Cao
- Department of Molecular and Cell BiologySchool of Life Sciences and TechnologyTongi UniversityShanghaiChina
| | - Changlin Mei
- Department of NephrologyKidney Institute of the People's Liberation ArmyChangzheng HospitalSecond Military Medical UniversityShanghaiChina
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38
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Perner B, Bates TJD, Naumann U, Englert C. Function and Regulation of the Wilms' Tumor Suppressor 1 (WT1) Gene in Fish. METHODS IN MOLECULAR BIOLOGY (CLIFTON, N.J.) 2018; 1467:119-28. [PMID: 27417964 DOI: 10.1007/978-1-4939-4023-3_10] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The Wilms' tumor suppressor gene Wt1 is highly conserved among vertebrates. In contrast to mammals, most fish species possess two wt1 paralogs that have been named wt1a and wt1b. Concerning wt1 in fish, most work so far has been done using zebrafish, focusing on the embryonic kidney, the pronephros. In this chapter we will describe the structure and development of the pronephros as well as the role that the wt1 genes play in the embryonic zebrafish kidney. We also discuss Wt1 target genes and describe the potential function of the Wt1 proteins in the adult kidney. Finally we will summarize data on the role of Wt1 outside of the kidney.
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Affiliation(s)
- Birgit Perner
- Leibniz Institute for Age-Fritz Lipmann Institute, Beutenbergstrasse 11, 07745, Jena, Germany
| | - Thomas J D Bates
- Leibniz Institute for Age-Fritz Lipmann Institute, Beutenbergstrasse 11, 07745, Jena, Germany
| | - Uta Naumann
- Leibniz Institute for Age-Fritz Lipmann Institute, Beutenbergstrasse 11, 07745, Jena, Germany
| | - Christoph Englert
- Leibniz Institute for Age-Fritz Lipmann Institute, Beutenbergstrasse 11, 07745, Jena, Germany. .,Friedrich Schiller University, Fürstengraben 1, 07743, Jena, Germany.
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39
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Drummond BE, Wingert RA. Scaling up to study brca2: the zeppelin zebrafish mutant reveals a role for brca2 in embryonic development of kidney mesoderm. CANCER CELL & MICROENVIRONMENT 2018; 5:e1630. [PMID: 29707605 PMCID: PMC5922780 DOI: 10.14800/ccm.1630] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Specialized renal epithelial cells known as podocytes are essential components of the filtering structures within the kidney that coordinate the process of removing waste from the bloodstream. Podocyte loss initiates many human kidney diseases as it triggers subsequent damage to the kidney, leading to progressive loss of function that culminates with end stage renal failure. Podocyte morphology, function and gene expression profiles are well conserved between zebrafish and humans, making the former a relevant model to study podocyte development and model kidney diseases. Recently, we reported that whole genome sequencing of the zeppelin (zep) zebrafish mutant, which exhibits podocyte abrogation, revealed that the causative lesion for this defect was a splicing mutation in the breast cancer 2, early onset (brca2) gene. This was a surprising and novel discovery, as previous research on brca2/BRCA2 in a number of vertebrate animal models had not implicated an explicit role for this gene in kidney mesoderm development. Interestingly, the abrogation of the podocyte lineage in zep mutants was also accompanied by the formation of a larger interrenal (IR) gland, which is analogous to the adrenal gland in mammals, and suggested a fate switch between the renal and inter renal mesodermal derivatives. Mirroring these findings, knockdown of brca2 also recapitulated the loss of podocytes and increased IR population. In addition, brca2 overexpression was sufficient to partially rescue podocytes in zep mutants, and induced ectopic podocyte formation in wild-type embryos. Interestingly, immunofluorescence studies indicated that zep mutants had elevated P-h2A.X levels, suggesting that DNA repair is dysfunctional in these animals and contributes to the zep phenotype. Moving forward, this unique zebrafish mutant provides a new model to further explore how brca2 contributes to the development of tissues including the kidney mesoderm-roles which may have implications for renal diseases as well.
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Affiliation(s)
- Bridgette E Drummond
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN, 46556, USA
| | - Rebecca A Wingert
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN, 46556, USA
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40
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Desgrange A, Heliot C, Skovorodkin I, Akram SU, Heikkilä J, Ronkainen VP, Miinalainen I, Vainio SJ, Cereghini S. HNF1B controls epithelial organization and cell polarity during ureteric bud branching and collecting duct morphogenesis. Development 2017; 144:4704-4719. [PMID: 29158444 DOI: 10.1242/dev.154336] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2017] [Accepted: 11/03/2017] [Indexed: 12/16/2022]
Abstract
Kidney development depends crucially on proper ureteric bud branching giving rise to the entire collecting duct system. The transcription factor HNF1B is required for the early steps of ureteric bud branching, yet the molecular and cellular events regulated by HNF1B are poorly understood. We report that specific removal of Hnf1b from the ureteric bud leads to defective cell-cell contacts and apicobasal polarity during the early branching events. High-resolution ex vivo imaging combined with a membranous fluorescent reporter strategy show decreased mutant cell rearrangements during mitosis-associated cell dispersal and severe epithelial disorganization. Molecular analysis reveals downregulation of Gdnf-Ret pathway components and suggests that HNF1B acts both upstream and downstream of Ret signaling by directly regulating Gfra1 and Etv5 Subsequently, Hnf1b deletion leads to massively mispatterned ureteric tree network, defective collecting duct differentiation and disrupted tissue architecture, which leads to cystogenesis. Consistently, mRNA-seq analysis shows that the most impacted genes encode intrinsic cell-membrane components with transporter activity. Our study uncovers a fundamental and recurring role of HNF1B in epithelial organization during early ureteric bud branching and in further patterning and differentiation of the collecting duct system in mouse.
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Affiliation(s)
- Audrey Desgrange
- Sorbonne Universités, UPMC Université Paris 06, IBPS - UMR7622, F-75005 Paris, France .,CNRS, UMR7622, Institut de Biologie Paris-Seine (IBPS) - Developmental Biology Laboratory, F-75005 Paris, France
| | - Claire Heliot
- Sorbonne Universités, UPMC Université Paris 06, IBPS - UMR7622, F-75005 Paris, France.,CNRS, UMR7622, Institut de Biologie Paris-Seine (IBPS) - Developmental Biology Laboratory, F-75005 Paris, France
| | - Ilya Skovorodkin
- Faculty of Biochemistry and Molecular Medicine, Biocenter, University of Oulu; Laboratory of Developmental Biology, Biocenter Oulu and InfoTech, Department of Medical Biochemistry and Molecular Medicine, Oulu Center for Cell Matrix Research, 90220 Oulu, Finland
| | - Saad U Akram
- Center for Machine Vision Research and Signal Analysis (CMVS), University of Oulu, FIN-90014, Oulu, Finland
| | - Janne Heikkilä
- Center for Machine Vision Research and Signal Analysis (CMVS), University of Oulu, FIN-90014, Oulu, Finland
| | | | | | - Seppo J Vainio
- Faculty of Biochemistry and Molecular Medicine, Biocenter, University of Oulu; Laboratory of Developmental Biology, Biocenter Oulu and InfoTech, Department of Medical Biochemistry and Molecular Medicine, Oulu Center for Cell Matrix Research, 90220 Oulu, Finland
| | - Silvia Cereghini
- Sorbonne Universités, UPMC Université Paris 06, IBPS - UMR7622, F-75005 Paris, France .,CNRS, UMR7622, Institut de Biologie Paris-Seine (IBPS) - Developmental Biology Laboratory, F-75005 Paris, France
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Abstract
Congenital abnormalities of the kidney and urinary tract (CAKUT) are one of the leading congenital defects to be identified on prenatal ultrasound. CAKUT represent a broad spectrum of abnormalities, from transient hydronephrosis to severe bilateral renal agenesis. CAKUT are a major contributor to chronic and end stage kidney disease (CKD/ESKD) in children. Prenatal imaging is useful to identify CAKUT, but will not detect all defects. Both genetic abnormalities and the fetal environment contribute to CAKUT. Monogenic gene mutations identified in human CAKUT have advanced our understanding of molecular mechanisms of renal development. Low nephron number and solitary kidneys are associated with increased risk of adult onset CKD and ESKD. Premature and low birth weight infants represent a high risk population for low nephron number. Additional research is needed to identify biomarkers and appropriate follow-up of premature and low birth weight infants into adulthood.
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Affiliation(s)
- Stacy Rosenblum
- Department of Pediatrics/Neonatology, Children's Hospital of Montefiore/Einstein, Bronx, NY, USA
| | - Abhijeet Pal
- Department of Pediatrics/Nephrology, Children's Hospital of Montefiore/Einstein, Bronx, NY, USA
| | - Kimberly Reidy
- Department of Pediatrics/Nephrology, Children's Hospital of Montefiore/Einstein, Bronx, NY, USA.
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Naylor RW, Han HI, Hukriede NA, Davidson AJ. Wnt8a expands the pool of embryonic kidney progenitors in zebrafish. Dev Biol 2017; 425:130-141. [PMID: 28359809 DOI: 10.1016/j.ydbio.2017.03.027] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2016] [Revised: 03/24/2017] [Accepted: 03/25/2017] [Indexed: 01/15/2023]
Abstract
During zebrafish embryogenesis the pronephric kidney arises from a small population of posterior mesoderm cells that then undergo expansion during early stages of renal organogenesis. While wnt8 is required for posterior mesoderm formation during gastrulation, it is also transiently expressed in the post-gastrula embryo in the intermediate mesoderm, the precursor to the pronephros and some blood/vascular lineages. Here, we show that knockdown of wnt8a, using a low dose of morpholino that does not disrupt early mesoderm patterning, reduces the number of kidney and blood cells. For the kidney, wnt8a deficiency decreases renal progenitor growth during early somitogenesis, as detected by EdU incorporation, but has no effect on apoptosis. The depletion of the renal progenitor pool in wnt8a knockdown embryos leads to cellular deficits in the pronephros at 24 hpf that are characterised by a shortened distal-most segment and stretched proximal tubule cells. A pulse of the canonical Wnt pathway agonist BIO during early somitogenesis is sufficient to rescue the size of the renal progenitor pool while longer treatment expands the number of kidney cells. Taken together, these observations indicate that Wnt8, in addition to its well-established role in posterior mesoderm patterning, also plays a later role as a factor that expands the renal progenitor pool prior to kidney morphogenesis.
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Affiliation(s)
- Richard W Naylor
- Department of Molecular Medicine and Pathology, University of Auckland, Auckland 1142, New Zealand.
| | - Hwa In Han
- Department of Developmental Biology, Center for Critical Care Nephrology, University of Pittsburgh, Pittsburgh, PA 15213, USA.
| | - Neil A Hukriede
- Department of Developmental Biology, Center for Critical Care Nephrology, University of Pittsburgh, Pittsburgh, PA 15213, USA.
| | - Alan J Davidson
- Department of Molecular Medicine and Pathology, University of Auckland, Auckland 1142, New Zealand.
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Pronephric tubule formation in zebrafish: morphogenesis and migration. Pediatr Nephrol 2017; 32:211-216. [PMID: 26942753 DOI: 10.1007/s00467-016-3353-1] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/24/2015] [Revised: 02/09/2016] [Accepted: 02/11/2016] [Indexed: 01/14/2023]
Abstract
The nephron is the functional subunit of the vertebrate kidney and plays important osmoregulatory and excretory roles during embryonic development and in adulthood. Despite its central role in kidney function, surprisingly little is known about the molecular and cellular processes that control nephrogenesis. The zebrafish pronephric kidney, comprising two nephrons, provides a visually accessible and genetically tractable model system for a better understanding of nephron formation. Using this system, various developmental processes, including the commitment of mesoderm to a kidney fate, renal tubule proliferation, and migration, can be studied during nephrogenesis. Here, we discuss some of these processes in zebrafish with a focus on the pathways that influence renal tubule cell morphogenesis.
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44
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Drummond BE, Li Y, Marra AN, Cheng CN, Wingert RA. The tbx2a/b transcription factors direct pronephros segmentation and corpuscle of Stannius formation in zebrafish. Dev Biol 2017; 421:52-66. [PMID: 27840199 PMCID: PMC5955707 DOI: 10.1016/j.ydbio.2016.10.019] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2016] [Revised: 10/21/2016] [Accepted: 10/27/2016] [Indexed: 12/25/2022]
Abstract
The simplified and genetically conserved zebrafish pronephros is an excellent model to examine the cryptic processes of cell fate decisions during the development of nephron segments as well as the origins of associated endocrine cells that comprise the corpuscles of Stannius (CS). Using whole mount in situ hybridization, we found that transcripts of the zebrafish genes t-box 2a (tbx2a) and t-box 2b (tbx2b), which belong to the T-box family of transcription factors, were expressed in the caudal intermediate mesoderm progenitors that give rise to the distal pronephros and CS. Deficiency of tbx2a, tbx2b or both tbx2a/b reduced the size of the distal late (DL) segment, which was accompanied by a proximal convoluted segment (PCT) expansion. Further, tbx2a/b deficiency led to significantly larger CS clusters. These phenotypes were also observed in embryos with the from beyond (fby)c144 mutation, which encodes a premature stop codon in the tbx2b T-box sequence. Conversely, overexpression of tbx2a and tbx2b in wild-type embryos expanded the DL segment where cells were comingled with the adjacent DE, and also decreased CS cell number, but notably did not alter PCT development-providing independent evidence that tbx2a and tbx2b are each necessary and sufficient to promote DL fate and suppress CS genesis. Epistasis studies indicated that tbx2a acts upstream of tbx2b to regulate the DL and CS fates, and likely has other targets as well. Retinoic acid (RA) addition and inhibition studies revealed that tbx2a and tbx2b are negatively regulated by RA signaling. Interestingly, the CS cell expansion that typifies tbx2a/b deficiency also occurred when blocking Notch signaling with the chemical DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester). Ectopic activation of Notch in Tg(hsp70::Gal4; UAS::NICD)(NICD) embryos led to a reduced CS post heat-shock induction. To further examine the link between the tbx2a/b genes and Notch during CS formation, DAPT treatment was used to block Notch activity in tbx2a/b deficient embryos, and tbx2a/b knockdown was performed in NICD transgenic embryos. Both manipulations caused similar CS expansions, indicating that Notch functions upstream of the tbx2a/b genes to suppress CS ontogeny. Taken together, these data reveal for the first time that tbx2a/b mitigate pronephros segmentation downstream of RA, and that interplay between Notch signaling and tbx2a/b regulate CS formation, thus providing several novel insights into the genetic regulatory networks that influence these lineages.
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Affiliation(s)
- Bridgette E Drummond
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Yue Li
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Amanda N Marra
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Christina N Cheng
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Rebecca A Wingert
- Department of Biological Sciences, Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556, USA.
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45
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46
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Abstract
The pronephros is the first kidney type to form in vertebrate embryos. The first step of pronephrogenesis in the zebrafish is the formation of the intermediate mesoderm during gastrulation, which occurs in response to secreted morphogens such as BMPs and Nodals. Patterning of the intermediate mesoderm into proximal and distal cell fates is induced by retinoic acid signaling with downstream transcription factors including wt1a, pax2a, pax8, hnf1b, sim1a, mecom, and irx3b. In the anterior intermediate mesoderm, progenitors of the glomerular blood filter migrate and fuse at the midline and recruit a blood supply. More posteriorly localized tubule progenitors undergo epithelialization and fuse with the cloaca. The Notch signaling pathway regulates the formation of multi-ciliated cells in the tubules and these cells help propel the filtrate to the cloaca. The lumenal sheer stress caused by flow down the tubule activates anterior collective migration of the proximal tubules and induces stretching and proliferation of the more distal segments. Ultimately these processes create a simple two-nephron kidney that is capable of reabsorbing and secreting solutes and expelling excess water-processes that are critical to the homeostasis of the body fluids. The zebrafish pronephric kidney provides a simple, yet powerful, model system to better understand the conserved molecular and cellular progresses that drive nephron formation, structure, and function.
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Affiliation(s)
- Richard W Naylor
- Department of Molecular Medicine and Pathology, School of Medical Sciences, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, 1142, New Zealand
| | - Sarah S Qubisi
- Department of Molecular Medicine and Pathology, School of Medical Sciences, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, 1142, New Zealand
| | - Alan J Davidson
- Department of Molecular Medicine and Pathology, School of Medical Sciences, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, 1142, New Zealand.
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Poureetezadi SJ, Cheng CN, Chambers JM, Drummond BE, Wingert RA. Prostaglandin signaling regulates nephron segment patterning of renal progenitors during zebrafish kidney development. eLife 2016; 5. [PMID: 27996936 PMCID: PMC5173325 DOI: 10.7554/elife.17551] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2016] [Accepted: 12/01/2016] [Indexed: 12/16/2022] Open
Abstract
Kidney formation involves patterning events that induce renal progenitors to form nephrons with an intricate composition of multiple segments. Here, we performed a chemical genetic screen using zebrafish and discovered that prostaglandins, lipid mediators involved in many physiological functions, influenced pronephros segmentation. Modulating levels of prostaglandin E2 (PGE2) or PGB2 restricted distal segment formation and expanded a proximal segment lineage. Perturbation of prostaglandin synthesis by manipulating Cox1 or Cox2 activity altered distal segment formation and was rescued by exogenous PGE2. Disruption of the PGE2 receptors Ptger2a and Ptger4a similarly affected the distal segments. Further, changes in Cox activity or PGE2 levels affected expression of the transcription factors irx3b and sim1a that mitigate pronephros segment patterning. These findings show for the first time that PGE2 is a regulator of nephron formation in the zebrafish embryonic kidney, thus revealing that prostaglandin signaling may have implications for renal birth defects and other diseases. DOI:http://dx.doi.org/10.7554/eLife.17551.001
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Affiliation(s)
- Shahram Jevin Poureetezadi
- Department of Biological Sciences, University of Notre Dame, Notre Dame, United States.,Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, United States
| | - Christina N Cheng
- Department of Biological Sciences, University of Notre Dame, Notre Dame, United States.,Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, United States
| | - Joseph M Chambers
- Department of Biological Sciences, University of Notre Dame, Notre Dame, United States.,Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, United States
| | - Bridgette E Drummond
- Department of Biological Sciences, University of Notre Dame, Notre Dame, United States.,Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, United States
| | - Rebecca A Wingert
- Department of Biological Sciences, University of Notre Dame, Notre Dame, United States.,Center for Stem Cells and Regenerative Medicine, Center for Zebrafish Research, University of Notre Dame, Notre Dame, United States
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Naylor RW, Dodd RC, Davidson AJ. Caudal migration and proliferation of renal progenitors regulates early nephron segment size in zebrafish. Sci Rep 2016; 6:35647. [PMID: 27759103 PMCID: PMC5069491 DOI: 10.1038/srep35647] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2016] [Accepted: 10/03/2016] [Indexed: 02/07/2023] Open
Abstract
The nephron is the functional unit of the kidney and is divided into distinct proximal and distal segments. The factors determining nephron segment size are not fully understood. In zebrafish, the embryonic kidney has long been thought to differentiate in situ into two proximal tubule segments and two distal tubule segments (distal early; DE, and distal late; DL) with little involvement of cell movement. Here, we overturn this notion by performing lineage-labelling experiments that reveal extensive caudal movement of the proximal and DE segments and a concomitant compaction of the DL segment as it fuses with the cloaca. Laser-mediated severing of the tubule, such that the DE and DL are disconnected or that the DL and cloaca do not fuse, results in a reduction in tubule cell proliferation and significantly shortens the DE segment while the caudal movement of the DL is unaffected. These results suggest that the DL mechanically pulls the more proximal segments, thereby driving both their caudal extension and their proliferation. Together, these data provide new insights into early nephron morphogenesis and demonstrate the importance of cell movement and proliferation in determining initial nephron segment size.
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Affiliation(s)
- Richard W Naylor
- Department of Molecular Medicine and Pathology, University of Auckland, Auckland 1142, New Zealand
| | - Rachel C Dodd
- Department of Molecular Medicine and Pathology, University of Auckland, Auckland 1142, New Zealand
| | - Alan J Davidson
- Department of Molecular Medicine and Pathology, University of Auckland, Auckland 1142, New Zealand
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Xu W, Jin M, Hu R, Wang H, Zhang F, Yuan S, Cao Y. The Joubert Syndrome Protein Inpp5e Controls Ciliogenesis by Regulating Phosphoinositides at the Apical Membrane. J Am Soc Nephrol 2016; 28:118-129. [PMID: 27401686 DOI: 10.1681/asn.2015080906] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2015] [Accepted: 05/30/2016] [Indexed: 12/23/2022] Open
Abstract
Phosphoinositides, a family of phosphorylated derivatives of phosphatidylinositol (PtdIns), are tightly regulated both temporally and spatially by PtdIns phosphatases and kinases. Mutations in inositol polyphosphate 5-phosphatase E (INPP5E) cause Joubert syndrome, a human disorder associated with numerous ciliopathic defects, including renal cyst formation, linking phosphoinositides to ciliopathies. However, the molecular mechanism by which INPP5E-mediated PtdIns signaling regulates ciliogenesis and cystogenesis is unclear. Here, we utilized an in vivo vertebrate model of renal cystogenesis to show that Inpp5e enzymatic activity at the apical membrane directs apical docking of basal bodies in renal epithelia. Knockdown or knockout of inpp5e led to ciliogenesis defects and cystic kidneys in zebrafish. Furthermore, knockdown of inpp5e in embryos led to defects in cell polarity, cortical organization of F-actin, and apical segregation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 Knockdown of the ezrin gene, which encodes an ezrin/radixin/moesin (ERM) protein that crosslinks PtdIns(4,5)P2 and F-actin, phenocopied inpp5e knockdowns. Notably, overexpression of the ezrin gene rescued inpp5e morphants. Finally, treatment with the PI 3-kinase inhibitor LY294002, which decreases PtdIns(3,4,5)P3 levels, rescued the cellular, phenotypic, and renal functional defects in inpp5e-knockdown embryos. Together, our data indicate that Inpp5e functions as a key regulator of cell polarity in the renal epithelia, by inhibiting PtdIns(3,4,5)P3 and subsequently stabilizing PtdIns(4,5)P2 and recruiting Ezrin, F-actin, and basal bodies to the apical membrane, and suggest a possible novel approach for treating human ciliopathies.
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Affiliation(s)
- Wenyan Xu
- Department of Molecular and Cell Biology, Tongji University School of Life Sciences and Technology, Shanghai, China
| | - Miaomiao Jin
- Department of Molecular and Cell Biology, Tongji University School of Life Sciences and Technology, Shanghai, China
| | - Ruikun Hu
- Department of Molecular and Cell Biology, Tongji University School of Life Sciences and Technology, Shanghai, China
| | - Hong Wang
- Department of Molecular and Cell Biology, Tongji University School of Life Sciences and Technology, Shanghai, China
| | - Fan Zhang
- Department of Molecular and Cell Biology, Tongji University School of Life Sciences and Technology, Shanghai, China
| | - Shiaulou Yuan
- Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut; and
| | - Ying Cao
- Department of Molecular and Cell Biology, Tongji University School of Life Sciences and Technology, Shanghai, China; .,Tongji University and Shanghai Changzheng Hospital Joint Research Center for Translational Medicine, Changzheng Hospital, Shanghai, China
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Omata M, Doke Y, Yamada C, Kawashima K, Sho R, Enomoto K, Furuya M, Inomata N. Hepatocyte Nuclear Factor-1β Induces Redifferentiation of Dedifferentiated Tubular Epithelial Cells. PLoS One 2016; 11:e0154912. [PMID: 27196561 PMCID: PMC4873210 DOI: 10.1371/journal.pone.0154912] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2016] [Accepted: 04/21/2016] [Indexed: 11/19/2022] Open
Abstract
Tubular epithelial cells (TECs) can be dedifferentiated by repetitive insults, which activate scar-producing cells generated from interstitial cells such as fibroblasts, leading to the accumulation and deposition of extracellular matrix molecules. The dedifferentiated TECs play a crucial role in the development of renal fibrosis. Therefore, renal fibrosis may be attenuated if dedifferentiated TECs are converted back to their normal state (re-epithelialization). However, the mechanism underlying the re-epithelialization remains to be elucidated. In the present study, TGF-β1, a profibrotic cytokine, induced dedifferentiation of cultured TECs, and the dedifferentiated TECs were re-epithelialized by the removal of TGF-β1 stimulation. In the re-epithelialization process, transcription factor hepatocyte nuclear factor 1, beta (HNF-1β) was identified as a candidate molecule involved in inducing re-epithelialization by means of DNA microarray and biological network analysis. In functional validation studies, the re-epithelialization by TGF-β1 removal was abolished by HNF-1β knockdown. Furthermore, the ectopic expression of HNF-1β in the dedifferentiated TECs induced the re-epithelialization without the inhibition of TGF-β/Smad signaling, even in the presence of TGF-β1 stimulation. In mouse renal fibrosis model, unilateral ureteral obstruction model, HNF-1β expression in the TECs of the kidney was suppressed with fibrosis progression. Furthermore, the HNF-1β downregulated TECs resulted in dedifferentiation, which was characterized by expression of nestin. In conclusion, HNF-1β suppression in TECs is a crucial event for the dedifferentiation of TECs, and the upregulation of HNF-1β in TECs has a potential to restore the dedifferentiated TECs into their normal state, leading to the attenuation of renal fibrosis.
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