Transcript Organizer gene activity

Developmental Biology – Biology 4361
Axis Formation and
Mesoderm Induction
October 27, 2005
Amphibian anteroposterior specification
- polarized eggs – animal/vegetal
- pigment
- yolk v. clear cytoplasm
- mitochondrial cloud
- germinal vesicle
- localized cytoplasmic components
RNA localization – Xenopus oocytes
Figure 8.25
Anteroposterior axis – VegT depletion
normal
Figure 9.7
Anteroposterior axis – VegT depletion
normal
VegT - depleted
depletion of VegT =
- shift from endoderm to mesoderm and ectoderm
- mesoderm replaced with ectoderm
- animal region forms only epidermis and no nervous system
Figure 9.7
Dorsalization Xenopus
UV = ventralized
Figure 9.18
Transplantation of dorsalizing activity
Figure 9.15
Early Dorsoventral Determination
Figure 9.19
Gray crescent formation
Cortical rotation and Disheveled
sperm
1. Fertilization
Disheveled
protein (Dsh)
2. Cortical rotation
Dsh
3. Dorsal enrichment
of Dsh
Disheveled activity
gylcogen synthase
kinase-3
Figure 9.21
Disheveled activity
gylcogen synthase
kinase-3
Transcription factor
Disheveled protein
blocks GSK-3 phosphorylation of b-catenin
Figure 9.21
Molecular basis of dorsoventral axis
b-catenin stabilized
b-catenin degraded
Tcf-3
proteins
b-catenin
siamois
proteins
gene
siamois
gene
Activated
Repressed
TGF-b
signaling
pathway
Siamois
protein
transcription
goosecoid
gene
transcription
Goosecoid
protein
Organizer transplant
Spemann’s organizer – dorsal lip of the blastopore
Organizer transplant
“Organizer” proteins
- expressed almost exclusively in the organizer
Nuclear Proteins
Secreted Proteins
XLim1
chordin
Xnot
noggin
Otx2
nodal-related proteins
(several)
XFD1
XANF1
Cerberus
Goosecoid
Follistatin
Frzb
Gilbert: Developmental Biology, 7th ed (2003) Table 10.2.
Organizer gene activity
goosecoid mRNA can induce a second dorsal axis:
goosecoid mRNA injection causes formation of a second dorsal blastopore lip
produces embryo with two dorsal axes and two sets of head structures
Gilbert: Developmental Biology, 7th ed (2003) Fig 10.28.
Organizer gene activity
Rescue of dorsal structures by noggin protein:
ventralized embryo without dorsal structures
(UV-irradiated)
dose-dependent induction of dorsal
structures by injection of noggin mRNA
“overdose” of noggin mRNA causes formation
of dorsal structures at the expense of
ventral structures
noggin binds to bone morphogenic proteins
(BMP2 & BMP4)
- inhibits binding BMP receptor binding
Gilbert: Developmental Biology, 7th ed (2003) Fig 10.30.
Organizer gene activity
chordin mRNA is localized in the ‘organizer’:
- chordin mRNA is found in the dorsal lip
- late in gastrulation, chordin is localized in the dorsal mesoderm of the notochord
- chordin protein binds to BMP4 and BMP2 – inhibits receptor BMP-receptor binding
- inhibition of BMP4 & BMP2 induces formation of the neural tube
in adjacent ectoderm
Gilbert: Developmental Biology, 7th ed (2003) Fig 10.32.
Mesoderm induction - Xenopus
Figure 9.8
Mesoderm induction - Xenopus
Figure 9.8
Mesoderm induction - Xenopus
Figure 9.8
Mesoderm induction - Xenopus
mesoderm inducers:
Vg1
bFGF
activin
Figure 9.9a, b
Mesoderm induction, Organizer formation
β-catenin
VegT, Vg1
Nodal
related
high
BMP4
low
Organizer
Nodal
related
low
BMP4
high
Ventral
mesoderm
1. β-catenin acts with VegT and Vg1 to activate Xnr genes (Xenopus Nodal-related)
2. Organizer originates in the region where VegT & Vg1 and β-catenin overlap
3. Gradient of Xnr protein specifies mesoderm: low Xnr  ventral mesoderm
4. High Xnr levels activate goosecoid and other ‘organizer’ genes
Left-right asymmetry
Most animals are bilaterally symmetrical (Bilateria)
- however, individuals deviate to some degree from true bilateral symmetry:
- fluctuating asymmetry: non-heritable minor left-right differences
- antisymmetry: heritable morphological left-right differences
- sidedness is randomly distributed (ca. 50% each)
- regular asymmetry or directed asymmetry:
sidedness is fixed for a species or for a higher taxon
e.g. in humans:
- heart on left side
- stomach curves to the left
- liver & spleen on right side
Left-right asymmetry
Deviation from directed asymmetry is often lethal!
- situs inversus: complete reversal of left-right symmetry in all organs
- heterotaxis: some organs reversed
- isomerism: normally asymmetrical organs duplicated or missing
Left-Right Asymmetry
Mechanistic basis for establishing asymmetry:
- chiral molecules may cause “symmetry-breaking” event
(specific orientation of stereoisomeric molecules relative to the body axes)
- translated into left-right differences at the level of cells, tissues
and the whole organism
Candidate chiral molecule: Dynein
- motor protein complex associated with axonemes, cilia
Left-Right Asymmetry
Dyneins - microtubule-associated motor protein complexes
Axonemal dyneins:
Fig 2.7.
- chiral: curve clockwise (from base) = ‘handedness’
- mediate sliding between adjacent microtubules in cilia or flagella
- cause cilia to rotate in a specific direction (clockwise)
- monocilia (at Hensen’s node - mouse) generate oriented flow of signal molecules
to the left side of the embryo
- signal molecules activate or inhibit patterning genes on left side
Iv+ and Inv+
iv+: ‘situs inversus viscerum’
- iv protein is a left-right dynein
- iv-/iv- = no motility, no fluid flow
- randomized L-R asymmetry (lethal)
inv+: “inversion of embryonic turning”
- wild type & heterozygous embryos turn clockwise
- inv-/inv- turn counterclockwise in amniotic cavity
- 100 % of homozygotes for inv show situs inversus
- mechanism of inv action is unknown
Nodal activated by iv,inv
Nodal
- intracellular protein - TGF-β family
- nodal gene activated by iv and inv genes
- nodal protein synthesized in left lateral plate mesoderm
- mesoderm adjacent to nodal expression develops into asymmetrical organs
- ectopic expression of nodal on right side randomizes location of the heart
- nodal is involved in determining left-right asymmetry in mice, frogs,
chicken & zebrafish
nodal expression in mouse:
wild type
ectopic
Pitx2 & lefty activated by iv, ivn, nodal
pitx2+ and lefty+ genes :
- pitx2 expression depends on iv, ivn and nodal genes
- pitx2 and lefty encode homeobox transcription factors that regulate genes
- both are expressed primarily on left side of vertebrate embryos
have been found in all vertebrates studied
- injection of ptx2 on right side of embryo
- can cause a complete reversal
of gut coiling and heart looping
nodal, pitx2 and lefty form an evolutionary
conserved signaling system that is
involved in regulating left-right
asymmetry in all vertebrates
pitx2 injection in Xenopus: