Developmental Biology and Regeneration
B.A. in English and B.S. in Biology, Muhlenberg College, Allentown, PA
Ph.D. in Cell and Developmental Biology, Harvard University, Cambridge, MA
Postdoctoral Research at Harvard Medical School and Massachusetts General Hospital, Boston, MA
OVERVIEW & SIGNIFICANCE:
My laboratory studies the mechanisms that direct organ development and regeneration. Currently, our primary focus is on the development and regeneration of the kidney. The kidney performs several essential physiological jobs. The kidney collects and excretes metabolic waste and also regulates water balance. In addition, the kidney produces hormones that regulate blood production and blood pressure. Because of these and other important functions, the loss of kidney activity has life-threatening consequences. Our long-term goals are to discover new biomedical interventions that can be used to treat or prevent kidney disease.
Kidney diseases represent a growing global healthcare burden: they affect epidemic numbers of children and adults worldwide, and are steadily climbing in incidence. Kidney diseases can be caused by congenital defects, acute and chronic injuries, malignancy, and as a secondary consequence of conditions like diabetes. Progressive damage to the kidney abolishes its functions and causes end-stage-renal disease (ESRD), which requires renal replacement therapy. Current renal replacement strategies can prolong life but have significant limitations: dialysis is grueling for patients, and there is a long wait (often >10 years) for an organ transplant. Taken together, there is a pressing need to identify innovative therapies to treat kidney disease and to identify renoprotective agents that could ameliorate and/or prevent kidney damage in various settings.
While kidney diseases are diverse in origin, many share a common trait: damage to the basic unit of the kidney called the nephron. Human kidneys vary in the number of nephrons they contain, ranging from several hundred thousand to over one million nephrons in a single kidney (Figure 1). Each nephron is an epithelial tube that is highly specialized. At one end of the nephron is a blood filter (or glomerulus) that interacts with the vasculature to collect fluid from the circulation. The collected filtrate then passes through a long tubule that consists of a series of different epithelial cell types, or so-called segments (indicated by colored intervals in Figure 1). Each tubule segment performs discrete tasks in modifying the filtrate by reabsorbing and secreting solutes—jobs that enable the retention of desirable nutrients and export of metabolic wastes. The last portion of the nephron connects to a collecting duct. Nephrons in mammalian kidneys have intricate loops and convolutions and are organized in arbor-like arrays, with the collected waste ultimately channeled into the bladder.
To date, the pathways that control how renal stem cells give rise to nephrons during kidney development are poorly understood. Part of the reason for this is that the architecture and internal location of the kidney pose challenges for studying nephron development and dysfunction in mammalian models like the mouse. Knowledge about how nephron cell types form during nephrogenesis would be a powerful tool in understanding congenital kidney defects and could be useful in the design of regenerative therapies for kidney diseases where nephrons (or particular cells within them) are destroyed.
For some time, there has been experimental evidence that the kidney exhibits a limited capacity to regenerate. Damage to epithelial cells in nephrons can be followed by a local regenerative response in which new nephron epithelial cells are made. However, the molecular pathways that enable this type of nephron regeneration (and others) remain a mystery and their discovery is hampered by the aforementioned limitations of existing mammalian models.
To study how nephrons are made during development and regenerate after injury, we use the zebrafish, Danio rerio. The zebrafish is an outstanding model for kidney research for numerous reasons. First, zebrafish are a vertebrate species and share many similarities with more complex vertebrates like mammals. For example, there is a high degree of conservation between gene function and basic cellular processes between zebrafish and mammals. Zebrafish embryos develop outside the mother and are optically transparent (Figure 2A), enabling the direct visualization of organ development. The embryo forms an anatomically simple kidney that is made up of two nephrons (Figure 2B). Zebrafish nephrons are comprised of segments with epithelial cells that share gene expression signatures and ultrastructural traits with segments in mammalian nephrons (indicated by the shared color in analogous segments between Figures 1 and 2B). Importantly, there is a diverse arsenal of molecular tools now available to the zebrafish researcher, and these enable high-resolution study of cell biology and genetic analysis.
Our research questions fall into two major categories:
(1) KIDNEY DEVELOPMENT RESEARCH: How do renal stem cells arise during development? How are nephrons constructed from renal stem/progenitor precursors? We are seeking to identify the genetic requirements for making renal cells. We perform genetic screens to isolate zebrafish with defects in nephron formation. One benefit of this approach is that we can discover essential genes and signaling pathways that have never been implicated in renal progenitor biology. In addition, we perform expression studies to identify factors expressed by kidney cells during nephrogenesis, then assign their functional roles using reverse genetics to knockdown the gene or overexpression techniques to examine gain-of-function.
(2) KIDNEY REGENERATION RESEARCH: How can damaged nephron components be replaced? Are there renal stem cells that can facilitate treatment of renal diseases? Can differentiated nephron cells be induced to regenerate damaged nephrons? Can the activation of kidney developmental pathways facilitate regeneration? We are using models of nephron injury in the embryo to discover the cell and molecular events that are required for nephron regeneration. We have designed a novel technique using laser ablation in which particular nephron regions are targeted for cell destruction, and are examining how such regions regenerate. Along with our nephron development studies, it is our hope that these lines of inquiry will shed novel insights into the activities of kidney cells and guide the creation of new therapeutics.
Ongoing research projects in the Wingert lab are supported by the Notre Dame Center for Zebrafish Research. We have received generous lab start-up funding from the University of Notre Dame College of Science and Department of Biological Sciences. We are funded currently by the National Institutes of Health (NIH) National Institute of Diabetes and Digestive and Kidney Diseases grant number K01DK083512, and the NIH Director’s New Innovator Award grant number DP2OD008470.
Figure 1: Composition of the mammalian kidney. (Left) The mammalian kidney is a bean-shaped organ comprised of functional units known as nephrons (enlarged in center). (Right) Nephrons have a segmental anatomy, being comprised of regions of specialized epithelial cells that perform discrete excretory
tasks. The major segments are indicated with different colors and segment names indicated. Abbreviations: PCT (proximal convoluted tubule), PST (proximal straight tubule), TAL (thick ascending limb), MD (macula densa), DCT (distal convoluted tubule), CD (collecting duct).
Figure 2: The zebrafish is a simple, conserved model in which to study kidney biology. (A) Lateral view of a zebrafish embryo two days after fertilization. The embryo is transparent, with limited pigmentation. (B) Lateral view schematic of the embryo, with the location of the kidney shown in purple. (Enlargement) Dorsal view of the embryonic kidney shows a pair of nephrons (top), with segmental anatomy indicated (bottom)
Johnson CS, Holzemer NF, Wingert RA. Laser ablation of the zebrafish pronephros to study renal epithelial regeneration. JoVE, 2011; 54. http://www.jove.com/details.php?id=2845, doi:10.3791/2845.
Gerlach GF, Schrader, LN, Wingert RA. Dissection of the adult zebrafish kidney. JoVE, 2011; 54. http://www.jove.com/details.php?id=2839, doi: 10.3791/2839.
O’Brien LL, Grimaldi M, Kostun Z, Wingert RA, Selleck R, Davidson AJ. Wt1a, Foxc1a, and the Notch mediator Rbpj physically interact and regulate the formation of podocytes in zebrafish. Dev Biol, 2011; 358(2): 318-30.
Wingert RA, Davidson AJ. Zebrafish nephrogenesis involves dynamic spatiotemporal expression changes in renal progenitors and essential signals from retinoic acid and irx3b. Dev Dyn, 2011; 240(8): 2011-2027.
Lengerke C*, Wingert RA*, Beeretz M, Grauer M, Schmidt AG, Konantz M, Daley GQ, Davidson AJ. Interactions between Cdx genes and retinoic acid modulate early cardiogenesis. Dev Biol, 2011; 354(1): 134-142. *equal co-first author contribution.
Diep CQ, Ma D, Deo RC, Holm TM, Naylor RW, Arora N, Wingert RA, Bollig F, Djordjevic G, Lichman B, Zhu H, Ikenaga T, Ono F, Englert C, Hukreiede NA, Handin RI, Davidson AJ. Identification of adult nephron progenitors capable of kidney regeneration in zebrafish. Nature, 2011; 470(7332): 95-100.
Fraenkel P, Gibert Y, Holzheimer JL, Lattanzi VJ, Burnett SF, Dooley, KA, Wingert RA, Zon LI. Transferrin-a modulates hepcidin expression in zebrafish embryos. Blood, 2009; 113(12): 2843-50.
Dooley KA, Fraenkel P, Langer NB, Schmid B, Davidson AJ, Weber G, Chiang K, Foott H, Dwyer C, Wingert RA, Zhou Y, Paw BH, Zon LI, Tübingen 2000 Screen Consortium. montalcino, a zebrafish model for variegate porphyria. Exp Hematol, 2008; 36(9): 1132-42.
Wingert RA and Davidson AJ. The zebrafish pronephros: a model to study segmentation. Kid Int, 2008; 73(10): 1120-1127.
Galloway JL, Wingert RA, Thisse C, Thisse B, and Zon LI. Combinatorial regulation of novel erythroid gene expression in zebrafish. Exp Hematol, 2008; 36(4): 424-432.
Wingert RA, Selleck R, Yu J, Song HD, Chen Z, Song A, Zhou Y, Thisse B, Thisse C, McMahon A, and Davidson AJ. The cdx genes and retinoic acid control the positioning and segmentation of the zebrafish pronephros. PLoS Genet, 2007; 3(10): e189.
Shaw GC, Cope JJ, Li L, Corson K, Hersey C, Ackermann GE, Gwynn B, Lambert AJ, Wingert RA, Traver D, Trede NS, Barut BA, Zhou Y, Minet E, Donovan A, Brownlie A, Balzan R, Weiss MJ, Peters LL, Kaplan J, Zon LI, and Paw BH. Mitoferrin is essential for erythroid iron assimilation. Nature, 2006; 440: 96-100.
Wingert RA, Zon LI. Genetic dissection of hematopoiesis using the zebrafish. In: Hematopoietic Stem Cells, Ed. Godin I, and Cumano A. 2006: 18-36. Georgetown: Texas: Landes Bioscience.
Wingert RA, Galloway JL, Barut B, Foott H, Fraenkel P, Axe J, Dooley K, Davidson AJ, Weber G, Paw B, Shaw G, Kingsley P, Palis J, Schubert H, Chen O, Kaplan J, Tübingen 2000 Screen Consortium, and Zon LI. glutaredoxin 5 deficiency reveals Fe/S clusters are required for vertebrate heme synthesis. Nature, 2005; 436: 1035-39.
Galloway JL, Wingert RA, Thisse C, Thisse B, and Zon LI. Loss of Gata1 but not Gata2 converts erythropoiesis to myelopoiesis in zebrafish embryos. Dev Cell, 2005; 8: 109-116.
Wingert RA, Brownlie A, Galloway JL, Dooley K, Fraenkel P, Axe J, Barut B, Davidson AJ, Noriega L, Sheng X, Zhou Y, and Zon LI. The chianti zebrafish mutant provides a model for erythroid-specific disruption of transferrin receptor 1. Development, 2004; 131: 6225-6235.