Iain A. Drummond (PI)
Mount Desert Island Biological Laboratory
To restore kidney function, filtering nephrons must connect to a tubule network to pass fluid. How cells rearrange and form new connections is not known. We are using the regenerating zebrafish kidney as a model to discover how tubule interconnections are made and to uncover signals that drive cell rearrangments required to "plumb" the kidney. Knowledge of these signals will be an important part of the molecular toolbox for growing new organs.
Liu, Yulong; Kossack, Michelle E; McFaul, Matthew E; Christensen, Lana N; Siebert, Stefan; Wyatt, Sydney R; Kamei, Caramai N; Horst, Samuel; Arroyo, Nayeli; Drummond, Iain A; Juliano, Celina E; Draper, Bruce W. eLife . 11:e76014. May 2022.
Zebrafish are an established research organism that has made many contributions to our understanding of vertebrate tissue and organ development, yet there are still significant gaps in our understanding of the genes that regulate gonad development, sex, and reproduction. Unlike the development of many organs, such as the brain and heart that form during the first few days of development, zebrafish gonads do not begin to form until the larval stage (≥5 days post-fertilization). Thus, forward genetic screens have identified very few genes required for gonad development. In addition, bulk RNA-sequencing studies that identify genes expressed in the gonads do not have the resolution necessary to define minor cell populations that may play significant roles in the development and function of these organs. To overcome these limitations, we have used single-cell RNA sequencing to determine the transcriptomes of cells isolated from juvenile zebrafish ovaries. This resulted in the profiles of 10,658 germ cells and 14,431 somatic cells. Our germ cell data represents all developmental stages from germline stem cells to early meiotic oocytes. Our somatic cell data represents all known somatic cell types, including follicle cells, theca cells, and ovarian stromal cells. Further analysis revealed an unexpected number of cell subpopulations within these broadly defined cell types. To further define their functional significance, we determined the location of these cell subpopulations within the ovary. Finally, we used gene knockout experiments to determine the roles of foxl2l and wnt9b for oocyte development and sex determination and/or differentiation, respectively. Our results reveal novel insights into zebrafish ovarian development and function, and the transcriptome profiles will provide a valuable resource for future studies.
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.
Djenoune, Lydia; Tomar, Ritu; Dorison, Aude; Ghobrial, Irene; Schenk, Heiko; Hegermann, Jan; Beverly-Staggs, Lynne; Hidalgo-Gonzalez, Alejandro; Little, Melissa H.; Drummond, Iain A.. Journal of the American Society of Nephrology . 32(7):1697–1712. July 2021.
Podocytes are critical to maintaining the kidney glomerular filtration barrier. Mutations in genes associated with development of nephrotic syndrome lead to elevated cytoplasmic calcium in podocytes and cause disruption of filtration barrier function. Whether calcium signaling plays a role in the initial formation of the filtration barrier is not known. Using live calcium imaging in two models, larval zebrafish and human kidney organoids, the authors demonstrate that podocyte calcium signaling is active during podocyte differentiation, is podocyte-cell autonomous, occurs independently of neighboring cell types, and is required for foot process and slit diaphragm formation. Their findings also show that developmental calcium signaling occurs by a different mechanism than disease-associated calcium perturbations, and represents a critical regulatory signal for podocyte morphogenesis and filtration barrier formation.Background Podocytes are critical to maintaining the glomerular filtration barrier, and mutations in nephrotic syndrome genes are known to affect podocyte calcium signaling. However, the role of calcium signaling during podocyte development remains unknown.Methods We undertook live imaging of calcium signaling in developing podocytes, using zebrafish larvae and human kidney organoids. To evaluate calcium signaling during development and in response to channel blockers and genetic defects, the calcium biosensor GCaMP6s was expressed in zebrafish podocytes. We used electron microscopy to evaluate filtration barrier formation in zebrafish, and Fluo-4 to detect calcium signals in differentiating podocytes in human kidney organoids.Results Immature zebrafish podocytes (2.5 days postfertilization) generated calcium transients that correlated with interactions with forming glomerular capillaries. Calcium transients persisted until 4 days postfertilization, and were absent after glomerular barrier formation was complete. We detected similar calcium transients in maturing human organoid glomeruli, suggesting a conserved mechanism. In both models, inhibitors of SERCA or IP3 receptor calcium-release channels blocked calcium transients in podocytes, whereas lanthanum was ineffective, indicating the calcium source is from intracellular podocyte endoplasmic-reticulum stores. Calcium transients were not affected by blocking heartbeat or by blocking development of endothelium or endoderm, and they persisted in isolated glomeruli, suggesting podocyte-autonomous calcium release. Inhibition of expression of phospholipase C-γ1, but not nephrin or phospholipase C-ε1, led to significantly decreased calcium activity. Finally, blocking calcium release affected glomerular shape and podocyte foot process formation, supporting the critical role of calcium signaling in glomerular morphogenesis.Conclusions These findings establish podocyte cell–autonomous calcium signaling as a prominent and evolutionarily conserved feature of podocyte differentiation and demonstrate its requirement for podocyte foot process formation.
Grainger, Stephanie; Nguyen, Nicole; Richter, Jenna; Setayesh, Jordan; Lonquich, Brianna; Oon, Chet Huan; Wozniak, Jacob M.; Barahona, Rocio; Kamei, Caramai N.; Houston, Jack; Carrillo-Terrazas, Marvic; Drummond, Iain A.; Gonzalez, David; Willert, Karl; Traver, David. Nature Cell Biology . 21(6):721–730. June 2019.
Wnt signalling drives many processes in development, homeostasis and disease; however, the role and mechanism of individual ligand–receptor (Wnt–Frizzled (Fzd)) interactions in specific biological processes remain poorly understood. Wnt9a is specifically required for the amplification of blood progenitor cells during development. Using genetic studies in zebrafish and human embryonic stem cells, paired with in vitro cell biology and biochemistry, we determined that Wnt9a signals specifically through Fzd9b to elicit β-catenin-dependent Wnt signalling that regulates haematopoietic stem and progenitor cell emergence. We demonstrate that the epidermal growth factor receptor (EGFR) is required as a cofactor for Wnt9a–Fzd9b signalling. EGFR-mediated phosphorylation of one tyrosine residue on the Fzd9b intracellular tail in response to Wnt9a promotes internalization of the Wnt9a–Fzd9b–LRP signalosome and subsequent signal transduction. These findings provide mechanistic insights for specific Wnt–Fzd signals, which will be crucial for specific therapeutic targeting and regenerative medicine.
Gallegos, Thomas F.; Kamei, Caramai N.; Rohly, Michael; Drummond, Iain A. Dev Biol . June 2019.
The zebrafish kidney regenerates after injury by development of new nephrons from resident adult kidney stem cells. Although adult kidney progenitor cells have been characterized by transplantation and single cell RNA seq, signals that stimulate new nephron formation are not known. Here we demonstrate that fibroblast growth factors and FGF signaling is rapidly induced after kidney injury and that FGF signaling is required for recruitment of progenitor cells to sites of new nephron formation. Chemical or dominant negative blockade of Fgfr1 prevented formation of nephron progenitor cell aggregates after injury and during kidney development. Implantation of FGF soaked beads induced local aggregation of lhx1a:EGFP + kidney progenitor cells. Our results reveal a previously unexplored role for FGF signaling in recruitment of renal progenitors to sites of new nephron formation and suggest a role for FGF signaling in maintaining cell adhesion and cell polarity in newly forming kidney epithelia.
Kamei, Caramai N.; Gallegos, Thomas F.; Liu, Yan; Hukriede, Neil; Drummond, Iain A. Development . 146(8). April 2019.
Zebrafish kidneys use resident kidney stem cells to replace damaged tubules with new nephrons: the filtration units of the kidney. What stimulates kidney progenitor cells to form new nephrons is not known. Here, we show that wnt9a and wnt9b are induced in the injured kidney at sites where frizzled9b- and lef1-expressing progenitor cells form new nephrons. New nephron aggregates are patterned by Wnt signaling, with high canonical Wnt-signaling cells forming a single cell thick rosette that demarcates: domains of cell proliferation in the elongating nephron; and tubule fusion where the new nephron plumbs into the distal tubule and establishes blood filtrate drainage. Pharmacological blockade of canonical Wnt signaling inhibited new nephron formation after injury by inhibiting cell proliferation, and resulted in loss of polarized rosette structures in the aggregates. Mutation in frizzled9b reduced total kidney nephron number, caused defects in tubule morphology and reduced regeneration of new nephrons after injury. Our results demonstrate an essential role for Wnt/frizzled signaling in adult zebrafish kidney development and regeneration, highlighting conserved mechanisms underlying both mammalian kidney development and kidney stem cell-directed neonephrogenesis in zebrafish.
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.