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Kidney Development: One variation on the theme of organogenesis Chris Campbell email@example.com 829-3462 Nov 6, 2012 Renal capsule Cross section of a human kidney Functions of the kidney Filters blood to remove toxins Regulates blood volume and composition The functional unit of the kidney is the nephron ~13,000 in mice ~1,000,000 in humans The functional unit of the kidney is the nephron Quaggin (2008) Devt 135, 609 Human Anatomy, 3rd edition, 2001 Techniques to Study Development • Observation of normal development in animals of different species • Reporter genes or other markers (such as fluoresceinated lectins or antibodies) to delineate particular cell types • Ablation and transplantation experiments • Use classical genetics to identify genes in which mutations affect kidney development or cause kidney cancer or other kidney disease. (phenotype to genotype) eg. Wt1 • Loss or gain of function mutations (knockouts, transgenics, injection of sense, antisense or small inhibitory RNAs) • In vitro organ and cell culture Similarities and Differences between Kidney Development and Lung Development Similarities •Involves inductive interactions between mesenchymal and epithelial cells •Branching morphogenesis •Stem cell maintenance and differentiation •Cell migration, oriented cell division and cell-ECM interactions Differences •Involves sequential formation of three pairs of increasingly complex organs, two of which are transient •Mesenchymal to epithelial transition Specification of the intermediate mesoderm. Koybayashi 2005 Dev 132, 2809 /Odd1 Dressler G R Development 2009;136:3863-3874 Development of the Kidney • The nephric duct and nephrogenic cord arise from intermediate mesoderm D>V • The nephric duct begins to elongate and undergo epithelialization in a rostral – caudal direction. (2010) Dev Cell 18, 698 Costantini ~E8.0 Laminin E-cadherin Bouchard Genes & Dev. 16,2958 (2002) •As this duct extends caudally (eventually joining up with the cloaca), the anterior region of the duct induces the adjacent mesenchyme to form the simple tubules of the pronephros . •In mammals this pronephros is non functional but in amphibian such as Xenopus it is a functional embryonic kidney consisting a single nephron. Development of the Urogenital System • (B)In mammals, the pronephric tubules and the anterior portion of the nephric duct degenerate, but the more caudal portions of the duct persist and serve as the Wolffian duct. • As the pronephric tubules degenerate, the middle portion of the nephric duct induces a new set of kidney tubules in the mesenchyme constituting the mesonephros or mesonephric kidney. Development of the Urogenital System • (C) In male mammals, some of the mesonephric tubules persist as the vas deferens and efferent ducts of the testes but the remainder degenerates. • The metanephric kidney is initiated by the outgrowth of the ureteric bud. ~ E10.5 rE14 Note rostral mesonephric tubules (arrows) are connected to the Wolfian Bouchard (2004) Differentiation 72, 295 duct (Wd) while the caudal tubules (arrowheads) are not. Immunostaining with anti brush border antigens Sainio et al. (1997) Dev 124, 1293 Development of the Metanephros • The development of the metanephros begins with the outgrowth of the ureteric bud from the Wolfian duct. • The ureteric bud grows out into the nephrogenic cord which then condenses around the bud to form the metanephric blastema or mesenchyme. • As this mesenchyme differentiates it induces the ureteric bud to branch and grow. • At the tips of the branches the mesenchyme undergoes epithelialization to form the structures of the nephron • The differentiated metanephric mesenchyme gives rise to the cells of the proximal and distal tubules, as well as the glomerular podocytes. Metanephric mesenchyme also gives rise to the renal stroma. • This process of branching of the UB and differentiation of the mesenchyme continues along a radial axis until ~P2-4 with the oldest nephrons located in the medulla and the newest nephrons in the periphery or nephrogenic zone. Shah et al (2004) Development 131, 1449 • The ureteric bud gives rise to the ureter, the renal pelvis, and the collecting duct system. Ureteric bud Cap tips mesenchyme E17.5 cap mesenchyme in Renal vesicle purple Pretubular aggregate http://www.gudmap.org/Organ_Summaries/Metanephros/index.html Comma shaped body S-shaped body Renal corpuscle Proximal tubule Loop of Henley Distal tubule Mature nephron vasculature Cortical collecting ducts Medullary collecting ducts Renal capsule Renal interstitium Kidney maturation involves reorientation within the body (Foxd1-/-) Levinson (2005) Dev 132, 529 Hatini (1996) Gen&Dev 10, 1467 Tbx18 E11.5 E14.5 +/+ -/- E18.5 Airik (2006) JCI 116, 663 In vitro organ culture Explanted murine kidney buds will partially differentiate in culture (epithelial and mesenchymal derived tissue only) Explanted kidney buds contained two morphologically different cell types (mesenchyme and ureteric bud) that can be mechanically or enzymatically separated. Saxen 1987 1. What happens when UB & metanephric mesenchyme are separated? Both undergo apoptosis. 2. What happens if they are separated and then put back together? Ureteric bud/metanephric mesenchyme co-cultures UB branches and mesenchyme forms tubules Miyamoto Dev. 124,1653 1997 Other Questions asked using UB/MM co-culture 1. What happens if they are separated by filters of increasingly small pore sizes? The smaller the pore size, the less differentiation is seen. Cells must be in contact 2. What happens if MM & UB are put together for some time and then separated? If MM is cultured with an inducer for 24 hours the inducer can then be removed and MM will contin to differentiate. Contact with UB necessary to initiate MM differentiation but not to mainta 3. Can an ‘induced’ MM induce a second MM? No. Induction of MM must be initiated by con with UB. 4. What happens if kidney epithelium or mesenchyme is replaced by epithelium or mesenchyme from another source? Ureteric bud or spinal cord (but not lung epithelium) will induce kidney mesenchyme to form Ureteric bud will not induce pulmonary mesenchyme to form tubules. Pulmonary mesenchyme will not induce ureteric bud to branch (unless the first branch has a occurred or GDNF is added to the medium). Kidney differentiation occurs as the result of reciprocal inductive interactions betwe mesenchyme and epithelium. At least some of these interactions require cell-cell co and for some of these interactions (but not all), either the ureteric bud (or the metan mesenchyme) can be replaced by another epithelium or mesenchyme. How do we identify genes important to kidney development? • Using reverse genetics to inactivate genes in mice a) with expression patterns that suggest they might play a role in kidney development or b) that are required for normal development in other species (e.g. Drosophila or human). (genotype to phenotype) eg. GDNF, Ret • Testing soluble molecules (including antisense oligonucleotides) or cell associated proteins in cell or organ culture. eg. GDNF or Wnt-4 • Using classical genetics to identify genes in which mutations affect kidney development or cause kidney cancer or other kidney disease. (phenotype to genotype) eg. Wt1 Odd1/Osr1 is the earliest known marker of IM E7.5 E8.5 E9.5 Wang (2005) Dev Biol 288, 582 Osr1eGFPCreERt2/+ x R26R Single tamoxifen injections from E6.5 – E 11.5 Harvest embryos at E15.5 and dual labeling b-gal and markers of specific kidney cell types. Mugford (2008) Dev Biol 324, 88 WT1 • WT1 was originally identified as a gene involved in Wilms tumor, a pediatric cancer in which kidney elements are incompletely differentiated and proliferate to form tumors. • Wt1 is first expressed in intermediate mesoderm prior to kidney development, and then in the kidney, gonads & mesothelium. • Based on the predicted sequence of the protein encoded by the Wt1 cDNA, Wt1 contained a DNA binding domain referred to as a “zinc finger” which implied Wt1 was likely a transcription factor. -/- The Wt1 Mouse no kidney E14.5 kidney E11.5 Wt1+/+ Wt1-/- In the absence of WT1 the UB fails to grow out from the Wolfian duct. Kreidberg et al. (1993) Cell 74, 679 The Wolfian duct in Wt1-/- mice is normal and can induce a wild type metanephric mesenchyme to differentiate normally but the mesenchyme in Wt1-/- mice cannot respond to signals from the UB. Donovan et al.(1999) Dev. Genet. 24, 252 At what stage in MM differentiaton are Wt1-/- mice defective ? Pax2 E11 -11.5 Donovan et al.(1999) Dev. Genet. 24, 252 Six2 Conclusion Even though Wt1 is expressed in the intermediate mesoderm prior to formation of the metanephric mesenchyme, the earliest stages of metanephric mesenchyme differentiation don’t require Wt1 expression (or contact with UB). Eya1 (MM) Xu (1999) Nat Gen 23, 113 Sall1 (MM) Emx2 E11.5 (UB) E11.5 Nishinakamura (2001) Dev 128, 3105 Miyamoto (1997) Dev 124, 1653 Six1 (MM) UB forms but fails to branch. Can be rescued in vitro with Grem1 (antagonist of BMP signaling). Nie (2011) Dev Bio 352, 141 Odd1 Ribes et al. J Am Soc Nephrol 14:S9, 2003 GDNF- ret Signaling in Kidney Development Majumdar (2003) Dev 130, 3175 • Ret was initially identified based on the ability of an oncogenic variant of the gene to transform NIH3T3 cells. • Sequence homology with other proteins identified ret as a receptor tyrosine kinase • Ret is expressed in the Wolfian duct and the ureteric bud. By the time the bud has branched several times, expression is restricted to the tips of the branches. Analyzing kidney -/- development in Ret Mice E11.5 E11.5 +/+ -/- • The phenotype of Ret-/- kidneys is variable ranging from complete absence of both kidneys and ureters to presence of two very small kidneys and relatively normal looking Schuchardt Nature 367,380 (1994) & Schuchardt (1996) Dev 122, 1919 ureters. -/- Analysis of Ret kidneys Conclusion (i)Mutant mesenchyme can signal wild type ureteric bud (ii) mutant ureteric bud cannot respond to wt mesenchyme. THE DEFECT IS IN THE URETERIC BUD. Schuchardt et al. (1996) Development 122, 1919 Identifying the ret receptor ligand GDNF was first identified as a factor capable of promoting growth of neurons in culture Following cloning of the gene, its expression pattern (expressed in MM but not UB) suggested it might play a role in kidney development. In organ culture contr +CM (conditioned medium from cells ol expressing GDNF) +aGDNF Ab +rhGDNF Vega et al. (1996) PNAS 93, 10657 GDNF-/- mice have kidney defects similar to but more severe than Ret-/- Pichel et al. (1996) Defects in enteric innervation and kidney development in mice lacking GDNF Nature 382, 73 Kidney from transgenic mouse expressing GDNF throughout the Wolfian duct. Spry1-/- Basson(2006) Dev Bio 299,466 Davidson 2009 stembook.org Rosen (2009) Wnt signaling and nephrogenesis Wnt4 E12.5 Wnt4 E11.5 E15 Wnt4-/- MM fails to undergo MET Stark (1994) Nature 372, 679 Isolated MM from either +/+ or Wnt4-/- mice can be induced to undergo tubulogenesis by Wnt Kispert (1998) Dev 125,4425 Wnt4 is required for MET of mesenchyme but Wnt4 is NOT expressed by UB. UB must e a protein that induces Wnt4 expression in mesenchyme. + Wnt9b>Wnt4>tubulogenesis (UB) (MM) Carroll (2005) Dev Cell 9,283 Six2 and the maintenance of nephron stem/progenitor cells E-cad (UB) Cad6 (nephron) Wt1 (cap mesenchyme) E11.5 kidney explants Overexpression of Six2 prevents MM differentiation Self(2006) EMBO25,5214 R26RlacZ b-gal- +cre b-gal+ Six2 expressing cells both self renewal & differentiate to contribute to all parts of th nephron (i.e. are stem cells). Wnt4 expressing cells differentiate but do not self rene Kobayashi (2008) Cell Stem Cell 3, 169 What made these investigators think FGF9 and FGF20 were candidates to maintain nephron progenitors? Why is it important that the Six2+ cell population that is maintained in culture by FGF9/FGF2 is still capable of differentiation? What would it mean if they weren’t? Why were some experiments performed with Fgf9+/-Fgf20-/- and some with Fgf9-/-Fgf20-/- double mutants? In discussing the pattern of expression of these two Fgfs the authors hypothesize that acts as a paracrine signal while Fgf20 acts as an autocrine signal. What does that mea how did they test this hypothesis? What did they conclude and why? Tha authors conclude that while loss of Fgf20 alone is not sufficient to cause bilateral r agenesis in humans, both Fgf9 and Fgf20 must be lost to cause a similar phenotype in Can you think of another possibility to explain their observation and how would you tes The authors state that in the absence of Fgf9 and Fgf20, nephron progenitors are forme not maintained. What is their evidence? What does it mean that the Six2+ cells couldn’t be maintained as long in culture as they normally live in vivo?
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