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