1. (10 pts) Beginning with gametogenesis and ending with birth, list the major stages of animal development. Describe the important defining features unique to each stage.

Developmental Biology 3230 31 Feb. 2003 Midterm Exam 1

1. (10 pts) Beginning with gametogenesis and ending with birth, list the major stages of animal development. Describe the important defining features unique to each stage.

GAMETOGENESIS

male gamete- haploid sperm (generally small with densely packed DNA and motile.

female gamete- haploid egg or oocyte (generally among the largest cells of the body, non motile)

FERTILIZATION

Mechanisms for assuring species specificity, block to polyspermy,

joining of genetic material from sperm and egg to produce zygotic nucleus and activates the program of embryogenesis.

CLEAVAGE

Stereotyped mitotic divisions whereby the large volume of the egg cytoplasm is parceled into many smaller cells. Stereotyped cleavage patterns may parcel out different cytoplasms and membranes(that contain different information molecules) into different blastomeres.

GASTRULATION

Gastrulation refers to the extensive rearrangement of cells that transforms the single layered epithelial bound cavity (blastocoel) into a three layered

structure. The three germ layers consist of the outer ectoderm (epidermis and nervous system), middle mesoderm (bones and muscles), and the inner endoderm (gut and associated organs).

ORGANOGENESIS

Through a process that often begins with induction cells multiply, migrate, differentiate, and organize themselves into organs.

BIRTH

Birth of most organisms marks the transition from development in the

controlled environment within the egg case or parent to the uncontrolled

cruel world.

2. (20 pts) Compare and contrast reproductive cloning to therapeutic cloning. Describe how each is accomplished. Give examples of where reproductive and therapeutic cloning could be beneficial. What are some of the arguments against both reproductive and therapeutic cloning?

Reproductive cloning is the process of creating a newborn individual who is genetically identical to another individual (human being). Therapeutic cloning is the process of creating stem cells that are genetically identical to an

individual for replacement of damaged cells. Both techniques are

accomplished by removing the nucleus of a matured egg and replacing it with a somatic nucleus (somatic nuclear transfer). The injected egg is exposed to a mixture of chemicals to activate development. In reproductive cloning the blastocyst is implanted into the uterus where it continues to develop into an individual. In therapeutic cloning stem cells are isolated from the blastocyst and cell lines are created that can be induced to differentiate into different cell types by exposure to different cell culture conditions.

Reproductive cloning would be beneficial to infertile couples that wish to have a child that inherits genetic material from the father or mother. There are also many animal applications. Therapeutic cloning has the potential of replacing damaged cells in a diseased individual (pancreatic islet cells, nerve cells, etc.)

Arguments against reproductive cloning include the risk to the mother, the low success rate of reproductive cloning (most clones die in utero), the risk of birth defects, and many other ethical considerations. Arguments against therapeutic cloning include the experimental use of embryonic stem cells that have the potential of developing into an individual human.

3. (15 pts) Compare the genome size of worms, flies, and humans.

How do these gene numbers relate to the number of cells and the apparent complexity of each organism? Suggests as many possibilities as you can to account for the apparent paradox concerning gene

number and organism complexity.

Worm- about 19,000 genes and 1000 somatic cells

Fly- about 14,000 genes and 100,000 cells (order of magnitude) Human- about 30,000 genes and 100 trillion cells (order of magnitude)

The sequence data does reveal some interesting differences between humans and simpler organisms. Humans have evolved a few new classes of protein domains (about 7%), and have expanded numbers of proteins involved in immune and nervous system functions. Other differences that may be

important in accounting for the differences in complexity are an increased use

of alternative splicing to make more “multitask” proteins, more complex post translational modifications (carbohydrates), and perhaps most important, larger and more complex regulatory domains. There is still some data that suggests 100,000 or more mRNA transcripts (EST data).

But others argue the large number of ESTs is just an artifact of the technique.

4. (10 pts) Describe the four main features of fertilization and the importance of each for ensuring normal development of the embryo.

1. Recognition events between sperm and egg: species specificity. If a sperm of the wrong species fertilized an egg it would most likely lead to embryo death or sterility.

2. Regulation of sperm entry into egg: block to polyspermy. If more than one sperm enters the egg it leads to aberrant spindle formation, chromosome assortment, and cleavages. The embryo normally dies

3. Fusion of sperm and egg genetic material. Necessary for regaining the diploid chromosome number, advantages of sex.

4. Activation of developmental process within the egg. Fertilization is tied to egg activation to assure the presence of a viable zygotic nucleus. If egg activates prematurely it generally leads to embryo death (although in some organisms you can parthanogenic reproduction).

5. (5 pts) Describe the functions of cortical granules in sea urchin fertilization.

The increased Ca concentration upon fertilization causes the cortical granules (about 15000 at 1 um) to fuse with the egg plasma membrane and release their contents. The enzymes released by the cortical granules inactivates the BINDIN receptors on the vitelline layer and a peroxidase cross links tyrosine residues of adjacent proteins within the vitelline layer so that sperm can no longer attach to or penetrate to the egg plasma membrane. Additionally attachments between the vitelline layer and the egg are digested releasing many osmotically active particles into the space between the vitelline and plasma membranes. Water rushes in and forces the vitelline layer away from the surface of the egg. It is now termed the FERTILIZATION ENVELOPE.

HYALIN from the cortical granules forms a protective and supportive protein

coat around the fertilized egg (now embryo).

6. (10) Fill in the missing terms in the 6 blanks

7. (10 pts) Compare and contrast the early development of the sea urchin and human embryo. Specifically address the differences and similarities in eggs and in the cleavage patterns (up to 5

cleavage).

Sea urchin and human eggs are isolecithal, although the sea urchin egg does contain more yolk. Both cleave holoblastically, but sea urchin cleaves with radial symmetry, while human second cleavage has rotational symmetry.

Cleavage in sea urchin is relatively rapid and regulated by maternal factors

while cleavage in humans is surprisingly slow (about 1/day) and controlled

zygotically. Fourth cleavage in sea urchins is unusual compared to human

because the animal hemisphere blastomeres cleave symmetrically in the

meridinal plane, while the vegetal pole blastomeres cleave asymmetrically in

the equatorial plane to produce the large vegetal macromeres and small

micromeres. Between 3

and 4

cleavage the human blastomeres undergo

compaction by expression of E-cadherin and formation of tight junctions.

8. (10) When do the first cell fate restrictions (or differences) arise in the sea urchin and mammalian embryo? Describe the experiments that were done to demonstrate that cells were no longer totipotent.

In sea urchins the first cell fate restriction occurs at the 3

cleavage that separates the blastomeres along the equatorial plane and creates the animal and vegetal blastomeres. (I will also give credit for formation of micromeres at 4

cleavage). In humans the first fate restriction occurs during compaction where the inner 2 or 3 cells will give rise to the inner cell mass and the outer cells are fated to give rise to the trophectoderm (extraembryonic tissues).

Isolation experiments demonstrate that the sea urchin animal blastomeres can no longer give rise to a complete embryo. Similar experiments with the trophectoderm cells demonstrate they are no longer totipotent. They can no longer contribute to all cell types if injected back into the inner cell mass.

9. (10) Compare the formation of the sea urchin and human gut(endoderm) during the first stages of gastrulation.

Primary mesenchyme are the first cells to ingress during sea urchin gastrulation. The vegetal plate of the sea urchin blastula that gives rise to endoderm first invaginates and then extends into the blastocoel by convergent extension. The final extension is via filopodia that pull the tip of the

archenteron to the side of the animal hemisphere where it induces the mouth.

Cellular ingression is the primary mechanism for endoderm formation in humans. Cells ingressing to form the endoderm are among the first to pass through the primitive groove during human gastrulation. The true endoderm is formed by cells ingressing through the primitive knot and migrating

anteriorly and deep to displace the hypoblast cells and form the foregut endoderm. Cells ingressing along the lateral lips of the primitive groove and migrating deep displace the hypoblast cells and give rise to the more posterior endoderm.

EXTRA CREDIT:

Describe the technique used in the SCID gene therapy trial. What problem arose and how did they try to explain it. Try to reconstruct the quantitative assumptions they must have made for their

explanation to be reasonable.