Todd Valerius (PI)
Brigham and Women's Hospital
Our objective is to aid RBK studies of in vitro cell populations by generating kidney cell type specific human pluripotent stem cell (hPSC) reporter lines. Such cell lines will enable both the optimization of differentiation protocols tailored to achieve specific cell types, and comparisons to in vivo human kidney cell types that will advance our understanding of in vitro tissue development and maintenance. We will use CRISPR/Cas9 gene editing to create GFP knock-in cell lines, targeting genes expressed in discrete cell types and according to consortium needs. These lines will be designed to label nephron and stromal progenitors, podocytes, and proximal and distal tubules. Additionally, cell type transition points offer glimpses into the factors that drive differentiation. We will create three dual-label reporter lines to illuminate transitions between progenitor and advanced cell types including nephron progenitors, fibrotic interstitium, and the juxtaglomerular apparatus.
Park, Joo-Seop; Ma, Wenxiu; O’Brien, Lori L.; Chung, Eunah; Guo, Jin-Jin; Cheng, Jr-Gang; Valerius, M. Todd; McMahon, Jill A.; Wong, Wing Hung; McMahon, Andrew P.. Dev Cell . 23(3):637–651. September 2012.
A balance between Six2-dependent self-renewal and canonical Wnt signaling-directed commitment regulates mammalian nephrogenesis. Intersectional studies using chromatin immunoprecipitation and transcriptional profiling identified direct target genes shared by each pathway within nephron progenitors. Wnt4 and Fgf8 are essential for progenitor commitment; cis-regulatory modules flanking each gene are cobound by Six2 and beta-catenin and are dependent on conserved Lef/Tcf binding sites for activity. In vitro and in vivo analyses suggest that Six2 and Lef/Tcf factors form a regulatory complex that promotes progenitor maintenance while entry of beta-catenin into this complex promotes nephrogenesis. Alternative transcriptional responses associated with Six2 and beta-catenin cobinding events occur through non-Lef/Tcf DNA binding mechanisms, highlighting the regulatory complexity downstream of Wnt signaling in the developing mammalian kidney.
Wiese, CB; Ireland, S; Fleming, NL; Yu, J; Valerius, MT; Georgas, K; Chiu, HS; Brennan, J; Armstrong, J; Little, MH; McMahon, AP; Southard-Smith, EM. Front Neurosci . 6:130. September 2012.
Relative positions of neurons within mature murine pelvic ganglia based on expression of neurotransmitters have been described. However the spatial organization of developing innervation in the murine urogenital tract (UGT) and the gene networks that regulate specification and maturation of neurons within the pelvic ganglia of the lower urinary tract (LUT) are unknown. We used whole-mount immunohistochemistry and histochemical stains to localize neural elements in 15.5 days post coitus (dpc) fetal mice. To identify potential regulatory factors expressed in pelvic ganglia, we surveyed expression patterns for known or probable transcription factors (TF) annotated in the mouse genome by screening a whole-mount in situ hybridization library of fetal UGTs. Of the 155 genes detected in pelvic ganglia, 88 encode TFs based on the presence of predicted DNA-binding domains. Neural crest (NC)-derived progenitors within the LUT were labeled by Sox10, a well-known regulator of NC development. Genes identified were categorized based on patterns of restricted expression in pelvic ganglia, pelvic ganglia and urethral epithelium, or pelvic ganglia and urethral mesenchyme. Gene expression patterns and the distribution of Sox10+, Phox2b+, Hu+, and PGP9.5+ cells within developing ganglia suggest previously unrecognized regional segregation of Sox10+ progenitors and differentiating neurons in early development of pelvic ganglia. Reverse transcription-PCR of pelvic ganglia RNA from fetal and post-natal stages demonstrated that multiple TFs maintain post-natal expression, although Pax3 is extinguished before weaning. Our analysis identifies multiple potential regulatory genes including TFs that may participate in segregation of discrete lineages within pelvic ganglia. The genes identified here are attractive candidate disease genes that may now be further investigated for their roles in malformation syndromes or in LUT dysfunction.
Yu, J; Valerius, MT; Duah, M; Staser, K; Hansard, JK; Guo, JJ; McMahon, J; Vaughan, J; Faria, D; Georgas, K; Rumballe, B; Ren, Q; Krautzberger, AM; Junker, JP; Thiagarajan, RD; Machanick, P; Gray, PA; van Oudenaarden, A; Rowitch, DH; Stiles, CD; Ma, Q; Grimmond, SM; Bailey, TL; Little, MH; McMahon, AP. Development . 139(10):1863–73. May 2012.
Lengthy developmental programs generate cell diversity within an organotypic framework, enabling the later physiological actions of each organ system. Cell identity, cell diversity and cell function are determined by cell type-specific transcriptional programs; consequently, transcriptional regulatory factors are useful markers of emerging cellular complexity, and their expression patterns provide insights into the regulatory mechanisms at play. We performed a comprehensive genome-scale in situ expression screen of 921 transcriptional regulators in the developing mammalian urogenital system. Focusing on the kidney, analysis of regional-specific expression patterns identified novel markers and cell types associated with development and patterning of the urinary system. Furthermore, promoter analysis of synexpressed genes predicts transcriptional control mechanisms that regulate cell differentiation. The annotated informational resource (www.gudmap.org) will facilitate functional analysis of the mammalian kidney and provides useful information for the generation of novel genetic tools to manipulate emerging cell populations.
Georgas, K; Rumballe, B; Valerius, MT; Chiu, HS; Thiagarajan, RD; Lesieur, E; Aronow, B; Brunskill, EW; Combes, AN; Tang, D; Taylor, D; Grimmond, SM; Potter, SS; McMahon, AP; Little, MH. Dev Biol . 332(2):273–86. August 2009.
While nephron formation is known to be initiated by a mesenchyme-to-epithelial transition of the cap mesenchyme to form a renal vesicle (RV), the subsequent patterning of the nephron and fusion with the ureteric component of the kidney to form a patent contiguous uriniferous tubule has not been fully characterized. Using dual section in situ hybridization (SISH)/immunohistochemistry (IHC) we have revealed distinct distal/proximal patterning of Notch, BMP and Wnt pathway components within the RV stage nephron. Quantitation of mitoses and Cyclin D1 expression indicated that cell proliferation was higher in the distal RV, reflecting the differential developmental programs of the proximal and distal populations. A small number of RV genes were also expressed in the early connecting segment of the nephron. Dual ISH/IHC combined with serial section immunofluorescence and 3D reconstruction revealed that fusion occurs between the late RV and adjacent ureteric tip via a process that involves loss of the intervening ureteric epithelial basement membrane and insertion of cells expressing RV markers into the ureteric tip. Using Six2-eGFPCre x R26R-lacZ mice, we demonstrate that these cells are derived from the cap mesenchyme and not the ureteric epithelium. Hence, both nephron patterning and patency are evident at the late renal vesicle stage.
Brunskill, EW; Aronow, B; Georgas, K; Rumballe, B; Valerius, MT; Aronow, B; Kaimal, V; Jegga, AG; Yu, J; Grimmond, SM; McMahon, AP; Patterson, LT; Little, MH; Potter, SS. Dev Cell . 15(5):781–91. November 2008.
Kidney development is based on differential cell-type-specific expression of a vast number of genes. While multiple critical genes and pathways have been elucidated, a genome-wide analysis of gene expression within individual cellular and anatomic structures is lacking. Accomplishing this could provide significant new insights into fundamental developmental mechanisms such as mesenchymal-epithelial transition, inductive signaling, branching morphogenesis, and segmentation. We describe here a comprehensive gene expression atlas of the developing mouse kidney based on the isolation of each major compartment by either laser capture microdissection or fluorescence-activated cell sorting, followed by microarray profiling. The resulting data agree with known expression patterns and additional in situ hybridizations. This kidney atlas allows a comprehensive analysis of the progression of gene expression states during nephrogenesis, as well as discovery of potential growth factor-receptor interactions. In addition, the results provide deeper insight into the genetic regulatory mechanisms of kidney development.
Little, MH; Brennan, J; Georgas, K; Davies, JA; Davidson, DR; Baldock, RA; Beverdam, A; Bertram, JF; Capel, B; Chiu, HS; Clements, D; Cullen-McEwen, L; Fleming, J; Gilbert, T; Herzlinger, D; Houghton, D; Kaufman, MH; Kleymenova, E; Koopman, PA; Lewis, AG; McMahon, AP; Mendelsohn, C; Mitchell, EK; Rumballe, BA; Sweeney, DE; Valerius, MT; Yamada, G; Yang, Y; Yu, J. Gene Expr Patterns . 7(6):680–99. June 2007.
Cataloguing gene expression during development of the genitourinary tract will increase our understanding not only of this process but also of congenital defects and disease affecting this organ system. We have developed a high-resolution ontology with which to describe the subcompartments of the developing murine genitourinary tract. This ontology incorporates what can be defined histologically and begins to encompass other structures and cell types already identified at the molecular level. The ontology is being used to annotate in situ hybridisation data generated as part of the Genitourinary Development Molecular Anatomy Project (GUDMAP), a publicly available data resource on gene and protein expression during genitourinary development. The GUDMAP ontology encompasses Theiler stage (TS) 17-27 of development as well as the sexually mature adult. It has been written as a partonomic, text-based, hierarchical ontology that, for the embryological stages, has been developed as a high-resolution expansion of the existing Edinburgh Mouse Atlas Project (EMAP) ontology. It also includes group terms for well-characterised structural and/or functional units comprising several sub-structures, such as the nephron and juxtaglomerular complex. Each term has been assigned a unique identification number. Synonyms have been used to improve the success of query searching and maintain wherever possible existing EMAP terms relating to this organ system. We describe here the principles and structure of the ontology and provide representative diagrammatic, histological, and whole mount and section RNA in situ hybridisation images to clarify the terms used within the ontology. Visual examples of how terms appear in different specimen types are also provided.
Naved, Bilal A.; Bonventre, Joseph V.; Hubbell, Jeffrey A.; Hukriede, Neil A.; Humphreys, Benjamin D.; Kesselman, Carl; Valerius, M. Todd; McMahon, Andrew P.; Shankland, Stuart J.; Wertheim, Jason A.; White, Michael J.V.; de Caestecker, Mark P.; Drummond, Iain A. Kidney International . March 2022.
Li, Li; Galichon, Pierre; Xiao, Xiaoyan; Figueroa-Ramirez, Ana C.; Tamayo, Diana; Lee, Jake J.-K.; Kalocsay, Marian; Gonzalez-Sanchez, David; Chancay, Maria S.; McCracken, Kyle W.; Lee, Nathan N.; Ichimura, Takaharu; Mori, Yutaro; Valerius, M. Todd; Wilflingseder, Julia; Lemos, Dario R.; Edelman, Elazer R.; Bonventre, Joseph V.. EMBO reports . 22(6):e51169. June 2021.
Recent studies demonstrate that metabolic disturbance, such as augmented glycolysis, contributes to fibrosis. The molecular regulation of this metabolic perturbation in fibrosis, however, has been elusive. COUP-TFII (also known as NR2F2) is an important regulator of glucose and lipid metabolism. Its contribution to organ fibrosis is undefined. Here, we found increased COUP-TFII expression in myofibroblasts in human fibrotic kidneys, lungs, kidney organoids, and mouse kidneys after injury. Genetic ablation of COUP-TFII in mice resulted in attenuation of injury-induced kidney fibrosis. A non-biased proteomic study revealed the suppression of fatty acid oxidation and the enhancement of glycolysis pathways in COUP-TFII overexpressing fibroblasts. Overexpression of COUP-TFII in fibroblasts also induced production of alpha-smooth muscle actin (αSMA) and collagen 1. Knockout of COUP-TFII decreased glycolysis and collagen 1 levels in fibroblasts. Chip-qPCR revealed the binding of COUP-TFII on the promoter of PGC1α. Overexpression of COUP-TFII reduced the cellular level of PGC1α. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potentially fibrosis in other organs.
Homan, Kimberly A.; Gupta, Navin; Kroll, Katharina T.; Kolesky, David B.; Skylar-Scott, Mark; Miyoshi, Tomoya; Mau, Donald; Valerius, M. Todd; Ferrante, Thomas; Bonventre, Joseph V.; Lewis, Jennifer A.; Morizane, Ryuji. Nature Methods . February 2019.
Kidney organoids derived from human pluripotent stem cells have glomerular- and tubular-like compartments that are largely avascular and immature in static culture. Here we report an in vitro method for culturing kidney organoids under flow on millifluidic chips, which expands their endogenous pool of endothelial progenitor cells and generates vascular networks with perfusable lumens surrounded by mural cells. We found that vascularized kidney organoids cultured under flow had more mature podocyte and tubular compartments with enhanced cellular polarity and adult gene expression compared with that in static controls. Glomerular vascular development progressed through intermediate stages akin to those involved in the embryonic mammalian kidney’s formation of capillary loops abutting foot processes. The association of vessels with these compartments was reduced after disruption of the endogenous VEGF gradient. The ability to induce substantial vascularization and morphological maturation of kidney organoids in vitro under flow opens new avenues for studies of kidney development, disease, and regeneration.
Oxburgh, L; Carroll, TJ; Cleaver, O; Gossett, DR; Hoshizaki, DK; Hubbell, JA; Humphreys, BD; Jain, S; Jensen, J; Kaplan, DL; Kesselman, C; Ketchum, CJ; Little, MH; McMahon, AP; Shankland, SJ; Spence, JR; Valerius, MT; Wertheim, JA; Wessely, O; Zheng, Y; Drummond, IA. J Am Soc Nephrol . 28(5):1370–1378. May 2017.
(Re)Building a Kidney is a National Institute of Diabetes and Digestive and Kidney Diseases-led consortium to optimize approaches for the isolation, expansion, and differentiation of appropriate kidney cell types and the integration of these cells into complex structures that replicate human kidney function. The ultimate goals of the consortium are two-fold: to develop and implement strategies for in vitro engineering of replacement kidney tissue, and to devise strategies to stimulate regeneration of nephrons in situ to restore failing kidney function. Projects within the consortium will answer fundamental questions regarding human gene expression in the developing kidney, essential signaling crosstalk between distinct cell types of the developing kidney, how to derive the many cell types of the kidney through directed differentiation of human pluripotent stem cells, which bioengineering or scaffolding strategies have the most potential for kidney tissue formation, and basic parameters of the regenerative response to injury. As these projects progress, the consortium will incorporate systematic investigations in physiologic function of in vitro and in vivo differentiated kidney tissue, strategies for engraftment in experimental animals, and development of therapeutic approaches to activate innate reparative responses.