Kidney Development

Chris Campbellcc59@buffalo.edu 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?