Chapter 10- Amphibians• Large yolks- hence, animal pole cleavage

dominates, while vegetal pole cleavage lags

1 2 3

4 micromeres + 4 macromeres (animal pole)4 macromeres (vegital pole)

Termed “morula”- 16-128 cells

4

5 8 Blastocoel

Cleavage

Fig. 10.1

GastrulationUse Xenopus as a model

Three tasks- 1. To bring inside those cells destined to be endodermal organs2. To surround embryo with cells destined for ectoderm3. To place cells cells “in between” destined to be mesoderm

Ectoderm-skin/nerves

Endoderm-Gut and associated organs

Mesoderm-Blood, heart, kidney,

bones

VegT establishes animal/vegital pole

VegT Antisense results in lack of endoderm

Fig. 10.6

Gastrulation

Gastrulation begins in marginal zone at dorsal lip, formed by bottle cells

Cells migrate toward dorsal lip, then turn inward into interior

Waves of cells continue to enter through dorsal lip

Animal pole cells migrate downward and eventually encase embryo, except for yolk plug,

termed epiboly (see fig. 8.6)

Blastocoel function1. permits cell migra-tion2. Prevents premature interactions

Animal

Vegital

Archenteron- formed by invading cells

Fig. 10.7

Gastrulation

Fig. 10.7

Order of cells entry through dorsal blastopore1. Pharengeal endomesoderm (foregut)2. Notochord and somite precursors3. Heart, kidney and ventral mesoderm

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

Sperm entry Dorsal blastopore lip formation

90 min0 min

30o

Fig. 7.35

Some details on Xenopus gastrulation

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

Of all tissues in early gastrula, only dorsal lip of blastopore has its cells fated

Transplant dorsal lip onto an embryo, observe a second invagination and a second embryo

Fig. 10.20

Thus, Dorsal lip cells dictate primary embryonic induction

The Spemann organizer

Evidence

Yeah, but what is the mechanism of axis formation by the Spemann Organizer??

Fig. 10.21

Answer- signals from vegetal cells

Normal “layers”Animal- EctodermMarginal- MesodermVegetal-Endoderm

If place animal cap next to vegetal cells- animal becomes mesoderm (muscle, etc)

Evidence

Vegetal cells that induce the Spemann organizer are called the Nieukoop center

Organizer

Nieukoop center

A mechanistic model of axis formation:

B-catenin accumulates in dorsal region to activate organizer genes, including goosecoid Fig. 10.23

B-catenin (orange)

How does B-catenin accumulate?Hint- It is initially throughout embryo.

Recall Wnt signaling pathway

from ch. 6

Fig. 6.23

Dishevelled expression inhibits GSK-3, thus stablezing B-catenen

A mechanistic model of axis formation:

Fig. 10.24

Dishevelled

Cortical rotation

Fertilization

Discheveled released

GSK-3 inhibited

B-catenin stabilized only in dorsal region

Siamois gene

B-catenin protein

Goosecoid gene Goosecoid

protein

TGF- signaling

B-catenin activates Samois expression, which activates Goosecoid expression

Organizer functions

• 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

The ability to

Dishevelled

GSK-3

B-catenin

samois

goosecoid

But what is goosecoid function??

-

-•If increase organizer mesoderm, observe expanded region of goosecoid expression•Inject goosecoid into 4-cell ventral blastomeres- progeny blastomeres induce cell migration and form dorsal lip

Activates two groups of proteins-

1. Bone morphogenesis protein (BMP) inhibitors2. Wnt inhibitors

Examples

Noggin, Chordin, Follistatin

Cerberus, FRZB, DICCK

Evidence of goosecoid’s importance

• Goosecoid also recruits neighboring cells into the dorsal axis

Thus, goosecoid must activate genes involved in dorsal/ventral and anterior/posterior axes

Example- Noggin (a BMP inhibitor)

If expose embryo to ultraviolet radiation- cortical rotation fails, and embryo lacks dorsal structures

How discovered?- cDNAs from a plasmid library injected into UV treated embryos.

Noggin mRNAinjected

Tail + belly

Normal

Head + belly

Only head

Fig. 10.29

Noggin functions by binding BMP4 and BMP2, thus preventing these proteins from binding their receptors

Chordin and follistatin also act by inhibiting BMP proteins

1. Bone morphogenesis protein (BMP) inhibitors

Example- Frzb (a Wnt inhibitor)

Fig. 10.36

How??Frzb functions by binding Xwnt8, thus preventing it from binding the wnt receptor

2. Wnt inhibitors

Frzb

Only head

Normal

Fig. 10.36

Fig. 10.35- Xwnt is inhibited by Frzb (produced by Organizer)

Thus, the Organizer secretes BMP and Wnt signaling inhibitors to direct head, belly and tail production

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 inductionThe 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 head2nd 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 developmentas 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?

•Vg1 protein activates nodal only on left side, which activates Pitx2

•Note that internal organs are not placed symmetrically- heart, gut tube