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