Amphibians and Fish: Early Development and Axis Formation BIOL 370 – Developmental Biology Topic #9 Amphibians and Fish: Early Development and Axis Formation Lange

Lazzaro Spallanzani – (1729 -1799) biologist and physiologist who made important contributions to the experimental study of bodily functions and animal reproduction.

Spallanzani’s most famous work examines the process of fertilization, and he mechanically isloated male gametes from female gametes and was able to induce fertilization in-vitro.

Various stages of development in the typical amphibian.

The Grey Crescent in frog eggs: Reorganization of the cytoplasm and cortical rotation produce the gray crescent in frog eggs The Grey Crescent in frog eggs: Due to the reorganization of the cytoplasm and rotation of the cortex In a) 50% of the cell cycle is complete, but no polarity In b) (70 % of the cell cycle complete) we see how the microtubules in the cells become parallel in the vegetal hemisphere. Together, these movements create the grey crescent. Note that the outer layer in black above (in C) is what we are referring to as the cortex.

Reorganization of the cytoplasm and cortical rotation produce the gray crescent in frog eggs (Part 2) DevBio9e-Fig-07-01-2R.jpg The gray crescent is a region of intermediate pigmentation where the first identifiable aspects of gastrulation will be seen. (two slides from now there is an even better rendition of this crescent.

Reorganization of the cytoplasm and cortical rotation produce the gray crescent in frog eggs (Part 3) Recall that the polarity of the cell is what is being identified here as the animal region (or animal pole) and the vegetal region (vegetal pole).

Cleavage of a frog egg DevBio9e-Fig-07-02-0.jpg

Scanning electron micrographs of frog egg cleavage Animal and vegetal pole cell size differences seen by the fourth division c). DevBio9e-Fig-07-03-0.jpg

Depletion of EP-cadherin mRNA in the Xenopus oocyte results in the loss of adhesion between blastomeres and the obliteration of the blastocoel “B” is showing the example of an embryo that lacks the EP-cadherin mRNA. The EP-cadherin (named because it appeared initially similar to both the E-cadherin and the P-cadherin) is required for adhesion in the blastomere Without these proteins, the formation of the blastocoel is not possible.

Standardized Color Scheme: Ectoderm – outer germ layer… will become nervous system, tooth enamel, epidermis, lining of the mouth, anus, nostrils, sweat glands, hair and nails. Mesoderm – middle germ layer… will become the muscle (smooth, cardiac and skeletal), connective tissues, dermis, hypodermis (subcutaneous layer of the skin), bone, cartilage, red blood cells, white blood cells, kidneys, and the adrenal cortex. Endoderm – inner germ layer… will become a variety of epithelia including the alimentary canal (excluding specialized parts of the mouth, pharynx & rectum), the lining cells of all the glands, trachea, bronchi, and alveoli of the lungs, endocrine glands, auditory tube, urinary bladder and parts of the urethra.

Cell movements during frog gastrulation DevBio9e-Fig-07-06-0.jpg I will split this diagram up to highlight specifics.

Cell movements during frog gastrulation (Part 1) Gastrulation is a phase early in the embryonic development of most animals, during which the single-layered blastula is reorganized into a trilaminar structure known as the gastrula. EARLY GASTRULATION DevBio9e-Fig-07-06-1R.jpg

Cell movements during frog gastrulation (Part 2) MID-GASTRULATION Identified by the formation of the archenteron which replaces the blastocoel. Note the development in orange, this endodermal tissue will become the BLASTOPORE. DevBio9e-Fig-07-06-2R.jpg

Cell movements during frog gastrulation (Part 3) X DevBio9e-Fig-07-06-3R.jpg Later Gastrulation…. note the elimination of the blastocoel.

Cell movements during frog gastrulation (Part 4) Final Stage of gastrulation….. the design is now called the GASTRULA. DevBio9e-Fig-07-06-4R.jpg

Surface view of an early dorsal blastopore lip of Xenopus In this Xenopus example, which side is the vegetal and which side is the animal region? Why did you select the positions you did? The animal pole is the side with the smaller cells and is in the upper region of this photographic image. The vegital pole is the portion with the larger cells and is in the lower part of this region. The “why” I am hoping to have you understand relates to the image a few slides ago where you could see distance cell size differences based upon polarity in the 16 cell stage. This is continuing through the point shown above.

Early movements of Xenopus gastrulation Focus on cell movement/migration that leads to the formation of the blastopore. DevBio9e-Fig-07-08-0.jpg

Epiboly of the ectoderm a cell movement that occurs in the early embryo, at the same time as gastrulation. It is one of many movements in the early embryo that allow for dramatic physical restructuring. Movement is characterized as being a thinning and spreading of cell layers. DevBio9e-Fig-07-09-0.jpg

Epiboly has been most extensively studied in zebrafish as their development allows for an easy visualization of the process. We will attempt to study zebrafish development in a lab nearer the end of the semester.

Xenopus gastrulation continues DevBio9e-Fig-07-10-0.jpg

Xenopus gastrulation continues (Part 1) The archenteron is the primitive gut that forms during gastrulation in the developing embryo is known as the archenteron. It develops into the digestive tract of an animal. DevBio9e-Fig-07-10-1R.jpg

The most common place you may have heard this term is in regard to the intercalated discs in cardiac muscle tissue.

Xenopus gastrulation continues (Part 2) Radial intercalation - part of the process of epiboly involves radial intercalation. Interior cells of the blastoderm move towards the outer cells, thus "intercalating" with each other. The blastoderm begins to thin as it spreads toward the vegetal pole of the embryo until it has completely engulfed the yolk cell. DevBio9e-Fig-07-10-2R.jpg To “intercalate” means to insert (something) between layers

Epiboly of the ectoderm is accomplished by cell division and intercalation DevBio9e-Fig-07-13-0.jpg

Spemann’s demonstration of nuclear equivalence in newt cleavage Hans Spemann’s work in 1903 demonstrated the concept of nuclear equivalence in this elegant experiment partially constricting the fertilized egg. The resultant development is associated with twinning. DevBio9e-Fig-07-14-0.jpg

Asymmetry in the amphibian egg Notice how normal development only proceeds when the cellular constriction occurs along the correct plane…. because the embryo is already asymetrical (as seen with the grey crescent). DevBio9e-Fig-07-15-0.jpg

Determination of ectoderm during newt gastrulation Notice how in the early gastrula the neural ectoderm transplant retains plasticity in development and becomes epidermis. By the time the embryo reaches the late gastrula stage this plasticity is lost. DevBio9e-Fig-07-16-0.jpg

Organization of a secondary axis by dorsal blastopore lip tissue Speeman & Mangold, in 1924 differentially colored embryos and then studied the organization of a secondary axis by transferring dorsal lip tissues. This further shows how a “twinning” may arise. DevBio9e-Fig-07-17-0.jpg

Transplantation and recombination experiments on Xenopus embryos Vegetal cells lying under the prospective blastopore lip begin gastrulation. DevBio9e-Fig-07-19-0.jpg Transplanting a slice of very dorsal vegetal cell in the 64-cell stage leads to twinning.

End.