Kidney Development
What does a mature kidney look like?
How does it develop?
Transcription factors required for kidney development : Wt1 & others
Signaling pathways involved: GDNF-ret & Wnt
Nephron stem/progenitor cells
Chris Campbell
cc59@buffalo.edu
829-3462
Oct 28, 2014
Renal capsule
Cross section of a human kidney
Functions of the kidney
The functional unit of the kidney is the nephron
~13,000 in mice
~1,000,000 in humans
Davidson 2009
The functional unit of the kidney is the nephron
Quaggin (2008) Devt 135, 609
Human Anatomy, 3rd edition, 2001
Kidney development begins with specification of the
intermediate mesoderm
/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.
Costantini (2010) Dev Cell 18, 698
Pax2 & Pax8 are required for the formation of the nephric duct
~E8.0
Laminin
E-cadherin
Bouchard Genes & Dev. 16,2958 (2002)
Development of the Pronephros
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 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
Bouchard (2004) Differentiation 72, 295
• (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.
~E10.5
Development of the Metanephros
•
•
•
•
•
•
•
Shah et al (2004) Development 131, 1449
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.
Branching of the ureteric bud
Watanabe 2004
Bohnenpoll 2014
Kidney maturation involves reorientation within the body
(Foxd1-/-)
Levinson (2005) Dev 132, 529
Foxd1
Pax2
Hatini (1996) Gen&Dev 10, 1467
Transcription factors and Metanephric Development
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 nephrogenic cord and MM
but not the WD or UB.
The Wt1-/- Mouse
E14.5
no kidney
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
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 mechanically
or enzymatically separated and then recombined. When separated both
tissues undergo apoptosis but if recombined UB will branch and MM will
differentiate into tubules
Inducer
Responder
Induction
Positive
Control
Wt1+/+
WD or UB
18/23
Negative
Controls
Wt1+/+ WD
Wt1+/+
Metanephric
mesenchyme
none
Wt1+/+
Metanephric
mesenchyme
Wt1-/Metanephric
mesenchyme
Wt1+/+
Metanephric
mesenchyme
3/42
none
Experiment Wt1+/+ WD
or UB
Wt1-/- WD
of MM differentiation
1/9
0/19
24/33
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 neither
signal 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
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).
Other transcription factors involved in kidney development
Eya1 (MM)
Xu (1999) Nat
Gen 23, 113
Sall1
(MM)
E11.5
Nishinakamura (2001) Dev 128, 3105
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
Emx2
(UB)
E11.5
Miyamoto (1997) Dev 124, 1653
Signaling Pathways and Kidney Development
Osr1
Ribes et al. J Am Soc Nephrol 14:S9, 2003
GDNF- Ret Signaling
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 ureters.
Schuchardt Nature 367,380 (1994) & Schuchardt (1996) Dev 122, 1919
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.
Mouse kidney organ culture
+ Conditioned medium from cells
expressing GDNF
control
+aGDNF
Ab
+recombinant human GDNF
Vega et al. (1996) PNAS 93, 10657
GDNF-/- mice have kidney defects similar to
(but more severe than) Ret-/-
Pichel et al. (1996) Nature 382, 73
Altering GDNF-ret signaling in any way affects affects kidney development
Spry1-/-
Basson(2006) Dev Bio 299,466
Rozen (2009) JASN 20, 255
Davidson 2009 stembook.org
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 Wnt4.
Kispert (1998) Dev 125,4425
UB expresses Wnt9b that induces Wnt4 expression in mesenchyme.
+
Wnt9b>Wnt4>tubulogenesis
(UB) (MM)
Carroll (2005) Dev Cell 9,283
Identifying renal stem/multipotent progenitor cells
Single MM cells from E11.5 expressing high levels of Sall1
(as measured by Sall1-GFP knock-in)
plated on 3T3Wnt4+ cells
can proliferate and differentiate into all
nephron cell types. (~10% of single cells plated).
Sall1 itself is NOT required (differentiation occurs in cells
isolated from Sall1-/- MM although colony size is reduced).
Osafune (2006) Dev 133, 151
Six2 and the identification of nephron stem/progenitor cells
(UB)
(nephron)
Wt1 (cap mesenchyme)
E11.5 kidney explants
Overexpression
of Six2 prevents
MM differentiation
Self(2006) EMBO25,5214
Formation of ectopic tubules in Six2-/- requires Wnt9b
Canonical Wnt signaling involves Wnt binding to its receptor which releases b-catenin from
a degradation complex. b-catenin levels rise, b-catenin enters nucleus and displaces a repression
complex (containing groucho proteins) bound to TCF/Lef. B-catenin/TCF acts as a transcriptional
activator inducing expression of target genes.
Park (2012) used Ch-IP/seq and transcription profiling to show that Six2 and Wnt (via
b-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 genes
require both Six2 and Wnt signaling to be expressed.
Main conclusion(s):
What is the next experiment?