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Developmental Biology – Biology 4361
Axis Formation andMesoderm Induction
October 27, 2005
Amphibian anteroposterior specification
- polarized eggs – animal/vegetal
- localized cytoplasmic components
- pigment
- yolk v. clear cytoplasm
- mitochondrial cloud
- germinal vesicle
Figure 8.25
RNA localization – Xenopus oocytes
Figure 9.7
Anteroposterior axis – VegT depletion
normal
Figure 9.7
depletion of VegT = - shift from endoderm to mesoderm and ectoderm - mesoderm replaced with ectoderm - animal region forms only epidermis and no nervous system
Anteroposterior axis – VegT depletion
normal VegT - depleted
Figure 9.18
Dorsalization - Xenopus
UV = ventralized
Figure 9.15
Transplantation of dorsalizing activity
Figure 9.19
Early Dorsoventral Determination
Gray crescent formation
Cortical rotation and Disheveled
sperm
Dsh
Disheveledprotein (Dsh)
1. Fertilization
2. Cortical rotation
3. Dorsal enrichment of Dsh
Figure 9.21
gylcogen synthasekinase-3
Disheveled activity
Figure 9.21
gylcogen synthasekinase-3
Disheveled protein
blocks GSK-3 phosphorylation of -catenin
Disheveled activity
Transcription factor
Molecular basis of dorsoventral axis
-catenin stabilized
Repressed
TGF-bsignalingpathway
-catenin degraded
Tcf-3proteins
siamoisgene
transcription
transcription
goosecoidgene
Goosecoidprotein
-cateninproteins
siamoisgene
Siamoisprotein
Activated
Organizer transplant
Spemann’s organizer – dorsal lip of the blastopore
Organizer transplant
Gilbert: Developmental Biology, 7th ed (2003) Table 10.2.
chordin
noggin
nodal-related proteins
(several)
XLim1
Xnot
Otx2
XFD1
XANF1
Goosecoid
Cerberus
Follistatin
Frzb
Secreted ProteinsNuclear Proteins
“Organizer” proteins
- expressed almost exclusively in the organizer
goosecoid mRNA can induce a second dorsal axis:
goosecoid mRNA injection causes formation of a second dorsal blastopore lip
Gilbert: Developmental Biology, 7th ed (2003) Fig 10.28.
Organizer gene activity
produces embryo with two dorsal axes and two sets of head structures
Rescue of dorsal structures by noggin protein:
Gilbert: Developmental Biology, 7th ed (2003) Fig 10.30.
“overdose” of noggin mRNA causes formation of dorsal structures at the expense of ventral structures
dose-dependent induction of dorsal structures by injection of noggin mRNA
ventralized embryo without dorsal structures (UV-irradiated)
Organizer gene activity
noggin binds to bone morphogenic proteins (BMP2 & BMP4) - inhibits binding BMP receptor binding
chordin mRNA is localized in the ‘organizer’:
Gilbert: Developmental Biology, 7th ed (2003) Fig 10.32.
Organizer gene activity
- inhibition of BMP4 & BMP2 induces formation of the neural tube in adjacent ectoderm
- chordin protein binds to BMP4 and BMP2 – inhibits receptor BMP-receptor binding
- late in gastrulation, chordin is localized in the dorsal mesoderm of the notochord
- chordin mRNA is found in the dorsal lip
Figure 9.8
Mesoderm induction - Xenopus
Figure 9.8
Mesoderm induction - Xenopus
Figure 9.8
Mesoderm induction - Xenopus
Figure 9.9a, b
Mesoderm induction - Xenopus
mesoderm inducers:
bFGF
Vg1
activin
Mesoderm induction, Organizer formation
β-catenin
VegT, Vg1
Nodalrelatedhigh
Nodalrelatedlow
Organizer
Ventralmesoderm
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
BMP4high
BMP4low
Left-right asymmetry
Most animals are bilaterally symmetrical (Bilateria)
- however, individuals deviate to some degree from true bilateral symmetry:
- 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
- fluctuating asymmetry: non-heritable minor left-right differences
- antisymmetry: heritable morphological left-right differences - sidedness is randomly distributed (ca. 50% each)
- 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
Deviation from directed asymmetry is often lethal!
Left-Right Asymmetry
Mechanistic basis for establishing asymmetry:
- translated into left-right differences at the level of cells, tissues and the whole organism
- chiral molecules may cause “symmetry-breaking” event
(specific orientation of stereoisomeric molecules relative to the body axes)
Candidate chiral molecule: Dynein
- motor protein complex associated with axonemes, cilia
Fig 2.7.
Dyneins - microtubule-associated motor protein complexes
- 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
Axonemal dyneins:
Left-Right Asymmetry
Iv+ and Inv+
- iv protein is a left-right dynein
- iv-/iv- = no motility, no fluid flow
- randomized L-R asymmetry (lethal)
iv+: ‘situs inversus viscerum’
- wild type & heterozygous embryos turn clockwise
inv+: “inversion of embryonic turning”
- inv-/inv- turn counterclockwise in amniotic cavity
- mechanism of inv action is unknown
- 100 % of homozygotes for inv show situs inversus
nodal expression in mouse:
Nodal activated by iv,inv
wild type ectopic
Nodal
- nodal is involved in determining left-right asymmetry in mice, frogs, chicken & zebrafish
- ectopic expression of nodal on right side randomizes location of the heart
- mesoderm adjacent to nodal expression develops into asymmetrical organs
- nodal protein synthesized in left lateral plate mesoderm
- nodal gene activated by iv and inv genes
- intracellular protein - TGF-β family
pitx2 injection in Xenopus:
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