Development of the Kidneys - Acsu Buffalo

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					       Kidney Development:
     One variation on the theme
         of organogenesis

Chris Campbell
                             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
• Observation of normal development in animals of different
• 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

    •Involves inductive interactions between mesenchymal and epithelial cells
    •Branching morphogenesis
    •Stem cell maintenance and differentiation
    •Cell migration, oriented cell division and cell-ECM interactions

    •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

Dressler G R Development 2009;136:3863-3874
Development of the Kidney
  • The nephric duct and
    nephrogenic cord arise
    from intermediate


  • The nephric duct begins
    to elongate and undergo
    epithelialization in a
    rostral – caudal direction. (2010) Dev Cell 18, 698


        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
              • (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
               • (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
                                          Note rostral mesonephric tubules
                                          (arrows) are connected to the
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
                                          •   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
                                          •   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

           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

     Levinson (2005) Dev 132, 529

Hatini (1996) Gen&Dev 10, 1467

         E11.5              E14.5



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
• 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 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


                   Wt1+/+                          Wt1-/-

In the absence of WT1 the UB fails to grow out from the Wolfian duct.

                                                       Kreidberg et al. (1993) Cell 74,
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 ?

                                                     E11 -11.5
                                     Donovan et al.(1999) Dev. Genet. 24, 252


 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

                                     (MM)                                         Emx2
                                     E11.5                                        (UB)

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

Ribes et al. J Am Soc Nephrol 14:S9, 2003
  GDNF- ret Signaling in Kidney
                                                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


•    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
      Analysis of Ret kidneys

Conclusion (i)Mutant mesenchyme can signal wild type ureteric bud
(ii) mutant ureteric bud cannot respond to wt

                 Schuchardt et al. (1996) Development 122, 1919
          Identifying the ret receptor
         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)

  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.


Basson(2006) Dev Bio 299,466                          Davidson 2009
                               Rosen (2009)
                            Wnt signaling and



                  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.

                                           (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

                                                          of Six2 prevents
                                                          MM differentiation
                                           Self(2006) EMBO25,5214



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

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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