Transcript Chapter 10- Amphibians

Cleavage
Chapter 10- Amphibians
• Large yolks- hence, animal pole cleavage
dominates, while vegetal pole cleavage lags
4 micromeres + 4 macromeres (animal pole)
4 macromeres (vegital pole)
1
5
2
3
4
8
Blastocoel
Fig. 10.1
Termed “morula”- 16-128 cells
Gastrulation
Use Xenopus as a model
Three tasks1. To bring inside those cells destined to be endodermal organs
2. To surround embryo with cells destined for ectoderm
3. To place cells cells “in between” destined to be mesoderm
Ectodermskin/nerves
VegT establishes
animal/vegital pole
MesodermBlood, heart, kidney,
bones
EndodermGut and associated organs
VegT Antisense results in lack
of endoderm
Fig. 10.6
Gastrulation
Cells migrate toward dorsal lip, then
turn inward into interior
Gastrulation begins in marginal zone at dorsal
lip, formed by bottle cells
Archenteron- formed by
Blastocoel
invading cells
Animal
function
1. permits
cell migraFig. 10.7
tion
2. Prevents
premature
interactions
Vegital
Waves of cells
Animal pole cells migrate downward and
continue to
eventually encase embryo, except for yolk plug, enter through
termed epiboly (see fig. 8.6)
dorsal lip
Gastrulation
Order of cells entry through dorsal blastopore
1. Pharengeal endomesoderm (foregut)
2. Notochord and somite precursors
3. Heart, kidney and ventral mesoderm
Fig. 10.7
Gastrulation
Some details on Xenopus gastrulation
Positioning the blastopore
•The point of sperm entry marks the ventral side
•The opposite side marks point of gastrulation initiation
•The cortical cytoplasm rotates 30 degrees toward sperm entry point
30o
Dorsal
Sperm entry
blastopore
lip
formation
Fig. 7.35
90 min
0 min
How do the invading cells know to crawl
along inside of animal pole?
Answer- fibronectin
•Cells on the blastocoel roof secrete
fibronectin, which invading mesodem
cells will track along
•Mesoderm receptors recognize the
Arg-Gly-Asp peptide
Evidence- if inject Arg-Gly-Asp
peptide, disrupt involution
Fig. 10.13
What dictates axis formation?
Answer- the Spemann
organizer – aka the dorsal
lip
Separate embryo
vertically
Separate embryo
horizontally
The dorsal lip is in the gray
crescent
Fig. 10.18
The Spemann organizer
Of all tissues in early
gastrula, only dorsal lip
of blastopore has its
cells fated
Evidence
Transplant dorsal
lip onto an
embryo, observe
a second
invagination and
a second embryo
Thus, Dorsal lip
cells dictate primary
embryonic induction
Fig. 10.20
Yeah, but what is the mechanism of axis formation by the
Spemann Organizer??
Answer- signals from vegetal cells
Normal “layers”
Animal- Ectoderm
Marginal- Mesoderm
Vegetal-Endoderm
Evidence
If place animal cap next to
vegetal cells- animal becomes
mesoderm (muscle, etc)
Vegetal cells that induce the
Spemann organizer are called
the Nieukoop center
Organizer
Nieukoop center
Fig. 10.21
A mechanistic model of axis formation:
B-catenin accumulates in dorsal
region to activate organizer
genes, including goosecoid
B-catenin (orange)
Fig. 10.23
How does B-catenin accumulate?
Hint- It is initially throughout embryo.
Recall Wnt
signaling pathway
from ch. 6
Dishevelled
expression inhibits
GSK-3, thus
stablezing B-catenen
Fig. 6.23
A mechanistic model of axis formation:
Fertilization
Cortical rotation
B-catenin activates Samois
Dishevelled expression, which activates
Goosecoid expression
B-catenin
Siamois
protein
gene
Discheveled released
GSK-3 inhibited
B-catenin stabilized
only in dorsal region
Fig. 10.24
TGF-b
signaling
Goosecoid
gene
Goosecoid
protein
Organizer functions
The ability to
• become dorsal mesoderm
• dorsalize surrounding ventral mesoderm
into lateral mesoderm
• dorsalize ectoderm into neural ectoderm
• initiate gastrulation movements
• cause neural plate to become neural tube
But what is goosecoid function??
Dishevelled Evidence of goosecoid’s importance
•If increase organizer mesoderm, observe
GSK-3
expanded region of goosecoid expression
•Inject goosecoid into 4-cell ventral
B-catenin
blastomeres- progeny blastomeres induce
samois
cell migration and form dorsal lip
• Goosecoid also recruits neighboring cells into the
goosecoid
dorsal axis
Thus, goosecoid must activate genes involved
in dorsal/ventral and anterior/posterior axes
Activates two
Examples
groups of proteins1. Bone morphogenesis
protein (BMP) inhibitors Noggin, Chordin, Follistatin
2. Wnt inhibitors
Cerberus, FRZB, DICCK
1. Bone morphogenesis protein (BMP) inhibitors
Example- Noggin (a BMP inhibitor)
How discovered?- cDNAs from a plasmid library injected into UV
treated embryos.
If expose embryo to ultraviolet radiationFig. 10.29
cortical rotation fails, and embryo lacks
dorsal structures
Tail + belly
Normal
Head + belly
Noggin functions by
binding BMP4 and
BMP2, thus preventing
these proteins from
binding their receptors
Chordin and follistatin also act by
inhibiting BMP proteins
Only head
Noggin mRNA
injected
2. Wnt inhibitors
Example- Frzb (a Wnt inhibitor)
Normal
Fig. 10.35- Xwnt is inhibited by
Frzb (produced by Organizer)
Only head
Fig. 10.36
Frzb
How??
Frzb functions by
binding Xwnt8, thus
preventing it from Thus, the Organizer secretes
BMP and Wnt signaling
binding the wnt
inhibitors to direct head, belly
receptor
and tail production
Fig. 10.36
Thus,
• BMP4 is the inducer of ventral ectoderm (epidermis)
• BMP4 is the ventralizer of mesoderm (connective tissue, blood)
• Noggin, chordin and follistatin (secreted by the Organizer)
inhibit BMP4
Regional specificity of induction
The organizer not only induced the neural tube, but also
specifies neural regions- forebrain, hindbrain,
spinocaudel region
1st organizer cells to enter embryo induce brain and head
2nd organizer cells become spinal cord and tail
Transplant
young
gastrula
dorsal lip
Transplant
advanced
gastrula
dorsal lip
Two heads
Two bellies?
What molecules are involved in regional specification?
Fibroblast growth factors (FGFs)•induces anterior neural tissue into posterior neural tissue
•Induces forebrain to express hind-brain-specific genes
Retinoic Acid (RA)•Overexpression inhibits fore
and mid-brain development
as wells as prevent posterior
structure formation
•Induces forebrain to express
hind-brain-specific genes
RA
3. What molecules are involved in left-right axis formation?
•Note that internal organs are not placed symmetrically- heart, gut tube
•Vg1 protein activates nodal only on left side, which activates Pitx2