Kidney Development
Chris [email protected] 829-3462 Oct 28, 2014
What does a mature kidney look like?How does it develop?Transcription factors required for kidney development : Wt1 & othersSignaling pathways involved: GDNF-ret & WntNephron stem/progenitor cells
Cross section of a human kidney
Functions of the kidney
Renal capsule
The functional unit of the kidney is the nephron
~13,000 in mice
~1,000,000 in humansDavidson 2009
The functional unit of the kidney is the nephron
Human Anatomy, 3rd edition, 2001Quaggin (2008) Devt 135, 609
Kidney development begins with specification of the intermediate mesoderm
Dressler G R Development 2009;136:3863-3874
/Odd1
Development of the Kidney• The nephric duct and
nephrogenic cord arise from intermediate mesoderm
Costantini (2010) Dev Cell 18, 698
D > V
• The nephric duct begins to elongate and undergo epithelialization in a rostral – caudal direction.
LamininE-cadherin
Bouchard Genes & Dev. 16,2958 (2002)
~E8.0
Pax2 & Pax8 are required for the formation of the nephric duct
As this duct extends caudally (eventually joining up with the cloaca), the anterior region of the duct induces the adjacent mesenchyme (NC) to form the simple tubule(s) of the pronephros .
•In mouse the pronephros consists of nothing more than a few
cell condensates and is non functional but in amphibians
such as Xenopus it is a functional kidney.
Chan (2006) Exp Neph 103 e81
• In mammals, 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.
Development of the Pronephros
Development of the Mesonephros
• (B) 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.
Sainio et al. (1997) Dev 124, 1293
Grote (2006) Dev 133, 53
Development of the Metanephros
• (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 from the Wolffian duct into the metanephric mesenchyme.
~E10.5Bouchard (2004) Differentiation 72, 295
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 metanephric mesenchyme 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 earliest nephrons located inside and the newest nephrons in the periphery or nephrogenic zone.
• The ureteric bud gives rise to the ureter, the renal pelvis, and the collecting duct system.
Shah et al (2004) Development 131, 1449
Watanabe 2004
Branching of the ureteric bud
Bohnenpoll 2014
Levinson (2005) Dev 132, 529
Kidney maturation involves reorientation within the body
Hatini (1996) Gen&Dev 10, 1467
(Foxd1-/-)
Foxd1 Pax2
• 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 nephrogenic cord and MM
but not the WD or UB.
Transcription factors and Metanephric Development
WT1
Wt1+/+ Wt1-/-
Kreidberg et al. (1993) Cell 74, 679
E14.5
E11.5
kidney
no kidney
The Wt1-/- Mouse
In the absence of WT1 the UB fails to grow out from the Wolfian duct.
Using in vitro organ culture to study the defect in WT1-/- mice
Explanted murine kidney buds will partially differentiate in culture.
Ureteric bud and metanephric mesenchyme can be mechanicallyor enzymatically separated and then recombined. When separated bothtissues undergo apoptosis but if recombined UB will branch and MM willdifferentiate into tubules
The Wolffian duct in Wt1-/- mice is normal and can induce a wild type metanephric mesenchyme to differentiate normally but the mesenchyme in Wt1-/- mice can neithersignal to the Wolffian duct to form a UB nor can it respond to signals from the UB.
Donovan et al.(1999) Dev. Genet. 24, 252
of MM differentiation
At what stage in MM differentiaton are Wt1-/- mice defective ?
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).
Pax2
Six2
E11 -11.5
Donovan et al.(1999) Dev. Genet. 24, 252
Emx2(UB)E11.5
Sall1(MM)E11.5
Eya1 (MM)Xu (1999) NatGen 23, 113
Nishinakamura (2001) Dev 128, 3105 Miyamoto (1997) Dev 124, 1653
Nie (2011) Dev Bio 352, 141
Six1 (MM) UB formsbut fails to branch. Can be rescued in vitro withGrem1 (antagonist of BMP signaling).
Other transcription factors involved in kidney development
Ribes et al. J Am Soc Nephrol 14:S9, 2003
Osr1Signaling Pathways and Kidney Development
GDNF- Ret Signaling
• 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.
Majumdar (2003)Dev 130, 3175
Analyzing kidney development in Ret-/- Mice
• 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 ureters.
Schuchardt Nature 367,380 (1994) & Schuchardt (1996) Dev 122, 1919
E11.5+/+
E11.5-/-
Analysis of Ret-/- kidneys
Schuchardt et al. (1996) Development 122, 1919
Conclusion (i)Mutant mesenchyme can signal wild type ureteric bud(ii) mutant ureteric bud cannot respond to wtmesenchyme. THE DEFECT IS IN THE URETERIC BUD.
Identifying the ret receptor ligand
control + Conditioned medium from cells expressing GDNF
+GDNFAb +recombinant human GDNF
Vega et al. (1996) PNAS 93, 10657
GDNF was first identified as a factor capable of promotinggrowth of neurons in cultureFollowing cloning of the gene, its expressionpattern (expressed in MM but not UB) suggested it might play a role in kidney development.
Mouse kidney organ culture
Pichel et al. (1996) Nature 382, 73
GDNF-/- mice have kidney defects similar to (but more severe than) Ret-/-
Altering GDNF-ret signaling in any way affects affects kidney development
Davidson 2009 stembook.orgBasson(2006) Dev Bio 299,466
Spry1-/-
Rozen (2009) JASN 20, 255
Wnt signaling and nephrogenesis
E12.5
E15
Stark (1994) Nature 372, 679
Kispert (1998) Dev 125,4425
Wnt4E11.5
Wnt4
Wnt4-/- MM fails to undergo MET
Isolated MM from either +/+ or Wnt4-/- mice can be induced to undergo tubulogenesis by Wnt4.
Wnt9b>Wnt4>tubulogenesis (UB) (MM)
Carroll (2005) Dev Cell 9,283
UB expresses Wnt9b that induces Wnt4 expression in mesenchyme.
+
Single MM cells from E11.5 expressing high levels of Sall1(as measured by Sall1-GFP knock-in) plated on 3T3Wnt4+ cellscan proliferate and differentiate into allnephron cell types. (~10% of single cells plated).Sall1 itself is NOT required (differentiation occurs in cellsisolated from Sall1-/- MM although colony size is reduced).
Osafune (2006) Dev 133, 151
Identifying renal stem/multipotent progenitor cells
Six2 and the identification of nephron stem/progenitor cells
(UB)(nephron)Wt1 (cap mesenchyme)
Overexpressionof Six2 preventsMM differentiation
Self(2006) EMBO25,5214
E11.5 kidney explants
Formation of ectopic tubules in Six2-/- requires Wnt9b
Park (2012) used Ch-IP/seq and transcription profiling to show that Six2 and Wnt (via -catenin & Tcf/Lef) regulate self-renewal and commitment of nephron progenitors through shared gene regulatory networks. Some genes activated by one are repressed by the other while other genesrequire both Six2 and Wnt signaling to be expressed.
Canonical Wnt signaling involves Wnt binding to its receptor which releases -catenin froma degradation complex. -catenin levels rise, -catenin enters nucleus and displaces a repressioncomplex (containing groucho proteins) bound to TCF/Lef. B-catenin/TCF acts as a transcriptionalactivator inducing expression of target genes.
Main conclusion(s): What is the next experiment?