Organizing centers: activators and inhibitors April 3, 2006

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Organizing centers activators and inhibitors
April 3, 2006
Didier Stainier
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Developmental Biology Journal Club (every other
Wednesday at 530 PM in Rock Hall room 302)
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symposium
dev
15th annual UCSF
bio
- 9th June 2006
Migration and Movement Marc Tessier-Lavigne Den
ise Montell Mark Krasnow John Wallingford Erez
Raz Ray Keller Scott Fraser
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The Question
1 cell type
200 different cell types
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Answer by the blastula stage (2000 cells)

• Evidence
• morphological differences
• fate map data
• e.g. ventral cells give rise to blood
• dorsal cells give rise to notochord
• - gene expression

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Answer at the blastula stage (2000 cells)

• Evidence
• morphological differences
• fate map data
• e.g. ventral cells give rise to blood
• dorsal cells give rise to notochord
• - gene expression

AP animal pole VP vegetal pole D dorsal V
ventral
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AP
V
D
VP
D
A
P
V
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Early developmental stages of Xenopus laevis
2.5 hpf
5 hpf
10 hpf
3.5 hpf
hpf hours post-fertilization
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Early developmental stages of Xenopus laevis
Cleavage movie
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Developmental stages of Xenopus laevis
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Gastrulation the ultimate cell migration problem
Danilchik movie
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Gastrulation and Neurulation highly
coordinated tissue movements
Keller neurulation movie
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Answer at the blastula stage (2000 cells)

• Evidence
• morphological differences
• fate map data
• e.g. ventral cells give rise to blood
• dorsal cells give rise to notochord
• - gene expression

AP animal pole VP vegetal pole D dorsal V
ventral
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The turning point of Hans Spemanns career
partial constriction
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Hilde Mangold and Hans Spemann, 1924
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Hilde Mangold and Hans Spemann, 1924
a
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Tissue from the dorsal side of the embryo
(dorsal lip of the blastopore) is transplanted
to the ventral side of the embryo.
This induces cells on the ventral side of the
embryo to form a second neuraxis ventrally
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Modern version of the Mangold and Spemann
experiment
Gastromaster movie http//www.xenbase.org/methods/
methods.html
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What is wrong with this representation of the
Mangold and Spemann experiment?
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A small region of the embryo, the organizer or
dorsal lip, has the ability when grafted to the
opposite (ventral) side to cause pronounced
cell non-autonomous effects. Neighboring cells
in the ectoderm are induced to form central
nervous system and those in the mesoderm to form
dorsal structures such as somites. These
inductive effects should be mediated by
diffusible molecules and the isolation of the
molecular signals involved has been a challenge
for generation of embryologists (Viktor
Hamburger, 1988).
Seeking the molecules responsible for the
activity of the Organizer (using the tools
available in late 1980s). a
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Seeking the molecules responsible for the
activity of the Organizer I. Inducing the
formation of an ectopic axis
Inject RNA (from Organizer) into
ventrovegetal blastomere
V
D
Lateral view
Formation of an ectopic axis
Dorsal view
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Seeking the molecules responsible for the
activity of the Organizer II. Rescuing UV
ventralized embryos
rescued embryo
UV ventralized embryo
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• Richard Harland (UC Berkeley)
• used an expression screen for activities that
  induce
• dorsal/head structures in Xenopus embryos
• - identified Noggin (Smith and Harland, Cell,
  1992)

• Eddy De Robertis (UCLA)
• used an expression screen for activities that
  rescue
• UV ventralized Xenopus embryos
• - identified Chordin (Sasai et al., Cell, 1994)

Two novel secreted proteins. What next?
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Data suggesting that Noggin and Chordin could
function by antagonizing BMP signaling.
BMPs act as ventralizers and are expressed in
the ventral side of the embryo (1992)
DN-BMP receptors have dorsalizing activity (1994)
The Drosophila homologue of Chordin
(Short-gastrulation) functions genetically as an
antagonist of Dpp (BMP homologue)
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Noggin and Chordin bind BMPs (BMP2, 4)
Zimmerman et al., Cell, 1996
Piccolo et al., Cell, 1996
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The model
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Additional members of the pathway were initially
identified genetically in Drosophila
Tolloid (clearly functions positively in BMP
signaling)
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Xolloid (Xenopus tolloid) cleaves Chordin,
reducing its affinity for BMP, thereby allowing
BMP binding to its receptor
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Additional members of the pathway were initially
identified genetically in Drosophila
Tolloid (clearly functioned positively in BMP
signaling)
Twisted gastrulation (function unclear) Initial
reports were confusing some claimed Tsg
promoted BMP signalling, the others claimed it
antagonized BMP signaling (Nature 2000, 2001)
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Genetic evidence for the Chordin/BMP model in
vertebrates?

• zebrafish
• mouse

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Zebrafish embryonic development
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Zebrafish embryos showing abnormal DV patterning
V ventralized
C dorsalized
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Zebrafish chordin mutants
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Chordin Noggin double mutant mouse embryos lack
the most anterior structures
wt
Chordin -/- Noggin /
Chordin -/- Noggin -/-
Bachiller et al., Nature, 2000
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Dorsal organizer transplant in zebrafish embryos
Saude et al., Development, 2000
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Organizer transplants in mouse embryos
Rosa Beddington
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Model of BMP4 gradient leading to different
mesodermal cell types
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What is responsible for the formation of
the dorsal organizer?
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Embryologically By 4 cell stage, the embryo is
asymmetric in its potential (Spemann, 1938)
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• Molecularly
• Wnt pathway implicated
• Wnt injection leads to secondary axis
• (Andy McMahon and Randy Moon, Cell, 1989)
• depletion of beta-catenin inhibits dorsal tissue
  formation
• (Janet Heasman, Chris Wylie, et al. Cell, 1994)
• Wnt11 appears to be the initiating event
• (Janet Heasman, Chris Wylie, et al. Cell, 2005)

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http//faculty.washington.edu/rtmoon/
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Wnt signaling, 2006 Xenopus assays, fly
genetics, yeast 2-hybrid
http//www.stanford.edu/rnusse/wntwindow.html
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http//faculty.washington.edu/rtmoon/cell2.html
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UV irradiation disrupts cortical MT and prevents
the transport of the membrane vesicles to the
prospective dorsal side
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Proteins secreted by the dorsal and ventral
signaling centers in the Xenopus gastrula
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Some of the functions of the dorsal organizer
(2000)
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What about in mouse? Is there early asymmetry?
Few maternal mRNAs are thought to be present in
the egg asymmetry thought to be coming from
extraembryonic tissues.
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• Fig. 1. Localization of Cdx2 (green) in mouse
  embryos by using an indirect immunofluorescence
  technique. White arrows indicate blastomeres with
  perinuclear localization of Cdx2. (A) Two-cell
  embryo with strong cytoplasmic localization of
  Cdx2 in one blastomere. The red arrow indicates a
  polar body (PB). (B) Typical pattern of Cdx2
  expression in a two-cell embryo. (C to E) Typical
  pattern of Cdx2 expression in three-, four-, and
  six-cell embryos. Scale bar indicates 25 µM.
• Deb et al. (2006). Science 311 992 - 996

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• Summary
• Key concepts
• Induction (organizing centers)

• Conservation of signaling pathways (vertebrates,
  fly)

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Conservation of mechanisms (DV patterning)
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• Summary
• Key concepts
• Induction (organizing centers)

• Conservation of signaling pathways (vertebrates,
  fly)

• Activators and Inhibitors

• Complexity

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Discussion paper
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http//anatomy.ucsf.edu/Pages/devbio/course.htm
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• http//www.nature.com/nrm/journal/v7/n4/extref/nrm
  1855-s1.avi (de Robertis doing the spemann
  transplant)
• The movie starts with photos of Hans Spemann and
  Hilde Mangold circa 1924 (Ref. 1). Next, it shows
  the author at the dissection microscope. Two
  embryos can be seen, one of which has the dorsal
  blastopore lip, the Spemann's organizer, clearly
  visible as a crescent. With the help of a
  tungsten needle and forceps, a square of
  organizer tissue is excised the operation is
  done free-hand. The organizer is pushed into the
  ventral side of a recipient gastrula with an
  eyebrow hair. One hour after transplantation, the
  graft has, almost miraculously, healed into the
  host embryo. Two days later, a Siamese twin has
  developed with two perfect body axes. The
  Spemann's organizer graft induced complete
  central nervous systems and mesodermal somites in
  tissues of the host that would otherwise have
  become ventral tissue.