Early Amphibian Development

Axis Formation

Determination of Amphibian Axes

• Arise progressively through a sequence of interactions between neighboring cells

• This is regulative development

– Where a isolated blastomere has a potency greater than its normal embryonic fate

– A cell’s fate is determined by interactions between neighboring cells - called inductions

• Such inductive interactions are responsible for amphibian axis determination

Early Experiments of Spemann

• Demonstrated early nuclear equivalence in newt cleavage (1903)

• He would lasso a a fertilized egg with a baby’s hair - this forced all the divisions to be on one side

• Once in awhile a nucleus would cross the the constriction into the non-nucleated side

• Cleavage would then also began on this side

• Tighten the lasso - separate the two halves - twin larvae develop, one a little older than the other

Spemann’s Demonstration - Nuclear Equivalence

Early Experiments of Spemann

• Second experiment - constriction this time was perpendicular to the original, but still longitudinal

• This time he left a region directly opposite to the sperm entry point, the gray crescent on one side

• The earlier experiment cut right through the gray crescent

Gray Crescent

– After fertilization the cortical cytoplasm shifts about 30° toward the point of sperm entry relative to the inner cytoplasm

– This exposes a region of the egg that was covered by dark cortical cytoplasm of the animal hemisphere

– The underlying cytoplasm has only diffuse pigment granules and appears gray - called the gray crescent

Reorganization of Cytoplasm after Fertilization

Early Experiments of Spemann

• 2nd Experiment

– The result was one normal embryo and one” belly piece”

– Thus something in the gray crescent was necessary for normal development

Asymmetry in the Amphibian Egg

Early Experiments of Spemann

• How does the gray crescent region function in normal development

• Answer from fate maps of the egg - gray crescent cells initiate gastrulation

– These cells form the dorsal lip of the blastopore

1918 Experiments of Spemann

• Early gastrula cells- not yet committed to a specific fate

– Said to exhibit conditional (regulative or dependent) development

– Fates dependent on location in the embryo

1918 Experiments of Spemann

• Late gastrula cells - when transplanted did not develop according to their new location - showed autonomous (independent or mosaic) development

– Their prospective fate was determined - cells developed independent of their new embryonic location

• A question needed to be answered.

– What was causing them to become determined?

Determination of Ectoderm During Gastrulation

Results of Tissue Transplantation - Gastrula

Hans Spemann & Hilde Mangold - 1924

• Only one tissue of the early gastrula has fate determined

– Dorsal lip of the blastopore (from gray crescent cytoplasm)

• Transplant this tissue in the presumptive belly region of a 2nd gastrula

• It stayed blastopore lip & also initiated gastrulation and embryogenesis - got two conjoined embryos instead of one

Dorsal Lip - Organization of a Secondary Axis

Dorsal Lip - Organization of a Secondary Axis

Hans Spemann & Hilde Mangold - 1924

• Spemann called the dorsal lip & their derivatives (notochord, prechordal mesoderm (become head mesoderm)) - organizer

• Organizer

1. Induced the host’s ventral tissues to change their fates to form neural tube & dorsal mesoderm (e.g. somites)

2. They organized host and donor tissues into a secondary embryo with clear anterior-posterior & dorsal-ventral axes

Organizer

• These cells organize the dorsal ectoderm into a neural tube & transform the flanking mesoderm into anterior-posterior body axis

• This is a key induction - all others depend upon it

– Called primary embryonic induction

Nieuwkoop Center

• Dorsal most vegetal cells of the blastula that are capable of inducing the organizer

• Demonstrated in 32 cell Xenopus embryo

• Gimlich and Gerhardt transplanted dorsalmost vegetal blastomere into the ventral vegetal side of another blastula

– Two embryonic axes were formed

Nieuwkoop Center

• Dale and Slack (1986) - recombined single vegetal blastomeres from a 32 cell stage Xenopus embryo with the upper-most animal tier of a fluorescently labeled embryo of the same stage

• The dorsalmost cells induced the animal pole to become dorsal mesoderm

• Remaining vegetal cells usually induce the animal cells to produce either intermediate or ventral mesodermal tissue

• Thus, dorsal vegetal cells can induce animal cells to become dorsal mesodermal tissue.

Molecular Biology - Nieuwkoop Center

• b-catenin appears to be responsible for forming the Nieuwkoop center

• b-catenin is necessary for forming the dorsal-ventral axis

Molecular Biology - Nieuwhoop Center

• How is b-catenin localized to the future dorsal cells of the blastula?

• Disheveled protein (associated with proteins in the vegetal pole) stabilizes b-catenin in the dorsal cells of the embryo

• Disheveled protein arrives there by cortical translocation during fertilization - thus disheveled protein moves to the dorsal side of the egg - via microtubules

• Now its released from its vesicles

• It can stabilize b-catenin by blocking GSK-3 from breaking it down

Model of Disheveled Protein Stabilizing b-catenin

Molecular Biology - Nieuwhoop Center

• Now b-catenin can complex with Tcf3 to form a transcription factor complex that can activate the siamois gene

• The siamois product & a TGF-b signal activate goosecoid gene in the organizer

• The goosecoid gene can activate other genes that bring about organizer function

Specifying Anterior-Posterior & Left-Right Axes

• Wnt3a, retinoic acid & eFGF are diffusible factors that influence anterior-posterior specification of the neural tube

• Left-right axis - initiated at fertilization via Vg1 protein

• Vg1 activates a Nodal protein - only on the left - this then activates expression of pitx2 gene

• pitx2 protein - critical in distinguishing left-sidedness from right-sidedness in the heart and gut