1

The Nephron

2

Yu et al., Curr. Opin. Gene. Dev. 14:550-7, 2004

3

Shah, M. M. et al. Development 2004;131:1449-1462

4

HoxB7:GFP

(ureteric bud

expression)

YesYesCombination

NoN.A.Metanephric

Mesenchyme

Alone

N.A.NoUreteric Bud

Alone

Tubules?Branching

Duct ?

Grobstein, 1955

Kidney Induction in Vitro

5

Many heterologous tissues can induce metanephric

mesenchyme to form tubules

But only the metanephric mesenchyme can form

tubules in response to inducers

Hence, the metanephric mesenchyme is highly

patterned before being induced to form tubules

6

Induction of the Ureteric Bud: C-ret and GDNF

C-ret: TK receptor expressed in the nephric duct and ureteric bud

C-ret -/- mice: No metanephros (although mesonephros is

normal)

Branching of the ureteric bud in tissue recombinations of c-ret -/-

and wild type tissues:

-+-/-

-++/+

-/-+/+

Bud

Mesenchyme Schuchardt et al.

Development 122,

1919-1929 (1996)

GDNF is a ligand for c-ret

GDNF -/- mice have no kidneys or

ureter

GDNF is expressed in the kidney

mesenchyme around the ureteric bud

branchpoints, and can induce ureteric

bud branching and outgrowth in vitro

(Sainio et al, Development 124, 4077-

4087, 1997)

7

Tissue-specific genes are expressed in the kidney mesenchyme

prior to its ineteraction with the duct

8

Pronephros

MesonephrosUreteric bud

Metanephros

Duct

Tubule

Glomus

Filtration

Barrier

The Vertebrate Kidney

som im

The Intermediate Mesoderm

Partial List of Intermediate Mesoderm Derivatives

Kidney, Somatic Gonad, Hematopoietic Tissue, Aorta, Adrenal

Glands

9

c

d coe

somlpn

ntB C D

n psm im lp

np

n som im lp

Asom im

10

11

Phase I:

Osr1

St. 9 St. 12

Phase II:

Pax2, lim1, wt1

Gene expression during intermediate mesoderm formation

A B C D

0h 5h 10h 15h

Fate Map of the Avian Intermediate Mesoderm

St. 5 St. 7 St. 9

12

im im

somsom somsom

nt

som

im im

lplp

A

D

B C

E F

quailpax-2

quailpax-2

IM is not specified in the primitive streak

som

nt

pax-2quail

imnt

som*

A CB

pax-2quail

im

lp

pax-2quail

im

lp lp

*

im

lp

*

D

IH

FE

G

IM becomes progressively specified as it migrates

from the primitive streak

IM is determined

with respect to

transplantation into

paraxial tissues by

St. 8

IM is NOT

determined with

respect to

transplantation into

lateral tissues at St.

8, but becomes

determined by St. 9

13

Paraxis(Mesogenin, Meox,

Tbx6)

Pax-2(Lim1, Wt1)

FoxC2 Osr1 Cytokeratin

Paraxial Paraxial/IM IM IM/LP LP

im

d coe

Som

(paraxial)

lpn

nt

Transcription Factors

in the mesoderm

Models of Intermediate Mesoderm Patterning:

The Medio-lateral Axis

Mediolateral gradient

Interaction between two already specified tissues

Vertical signaling

14

pax2

lim1

pax2

quail

Quail somite

Quail somite

+ Chick

lateral plate

mesoderm

A property in the lateral plate which can induce

IM genes in the somite

Bmp signaling at the time of initial Intermediate

Mesoderm formation

Bmp-2 Bmp-4 P-Smad1

P-Smad1 from Faure et al., Dev. Biol. 244:44 (2002)

15

Normal

Bmp

Noggin

osr1 lim1 wt1

b

b b

nn

Alterations in IM gene expression by manipulation of

Bmp signaling in vivo

b

n

b

n

c

c

c c

Bmp-2 can generate IM from paraxial mesoderm

in a dose-responsive manner

osr1 lim1 cytokeratin

St. 5-6

anterior

primitive

streak

St. 8

paraxial

mesoderm

paraxis

16

Does BMP regulate IM gene expression cell-

autonomously?

Bmp signaling can induce IM gene expression

cell-autonomously

pCS2-c.a.

Alk3-IRES-

GFP

Gfp Scl/Tal

Control

Gfp

Gfp

Pax2

Osr1

psm

17

Direct responders to Bmp signaling ?

Msx2 Osr1 Tbx6

No Rx

Bmp2

CHX

Bmp2 +

CHX

St. 8 PSM

cultured for 3

hours

Directly

activated

Not directly activated;

Under negative

regulation?

Not directly

repressed

Paraxis(Mesogenin, Meox,

Tbx6)

Pax-2(Lim1, Wt1)

FoxC2 Osr1 Cytokeratin

Paraxial Paraxial/IM IM IM/LP LP

im

d coe

Som

(paraxial)

lpn

nt

Transcription Factors

in the mesoderm

18

Intermediate Mesoderm markers are expanded

medially in FoxC1/C2 mutants

Osr1

Osr1

Lim1

FoxC2 represses Pax-2 in the IM

pMES-GFP

(Control)

pMES-FoxC2-

GFP

pMES-FoxC2-

GFP

Dapi Pax-2 Gfp

somim

somim

im im

im imim im

Pax-2 Gfp

Pax-2 Gfp

19

Untransfected

pMES-FoxC2-GFP

Dapi

Dapi

Pax-7

Pax-7

FoxC2 induces expression of the somite marker Pax-7 in the IM

Gfp

s ss

im im im

imim ss

osr1,pax,

lim,etc

FoxC

Pax7

Somite IM Lateral Plate

FoxC

Pax7

Bmp

FoxC

Pax7

Bmp

High

Bmp

Paraxis Pax-2 Osr1 Cytokeratin

CK

osr1,pax,

lim,etc

osr1,pax,

lim,etc

20