Developmental Biology - Exam 2

Front 1

Four major events of fertilization.

Contact and recognition between sperm and egg -

Back 1

Sperm are attracted towards eggs of their species by chemotaxis, following a gradient of chemicals secreted by the egg. Resact, which diffuses in seawater was isolated from egg jelly of the sea urchin. After a small amount of resact is injected into a drop of sea water containing sperm, the sperm migrate to the region of injection. Resact receptors are located on the sperm and sperm will swim up concentration gradients of resact to the egg.

Bindin, a protein on the acrosome process, is capable of binding to the eggs of the same species.

Front 2

Four major events of fertilization.

Regulation of sperm entry into egg.
Fast Block -

Back 2

Fast block to polyspermy is achieved by changing the electric potential of the eggs plasma membrane via a concentration gradient of Na+. Resting potential is -70mV. Within 3 seconds of binding with the first sperm, a Na+ influx changes the membrane potential to +20mV, and sperm cannot fuse with a membrane whose potential is (+). Polyspermy can be induced if sea urchin eggs are supplied with an electric current which keeps the membrane potential engative. Vice versa... Fast block may also be induced by lowering the concentratino of Na+ in the water.

Front 3

Four major events of fertilization.

Regulation of sperm entry into egg.
Slow Block -

Back 3

Slow block to polyspermy is achieved by cortical granule reaction. The cortical granule exocytose proteases which dissolve the protein posts connecting the vitelline envelope with the cell membrane and clip off bindin receptors. Peroxidase then hardens the fertilization envelope. Hyaline forms a coating around the egg. A release of calcium from intercellular storage initiates the cortical granule reaction (IP3 pathway initiated during slow block). When an egg is injected with a fluorescent dye and then fertilized, a band of light travels across the cell from the point of sperm entry.

--A23187, a calcium ionophore, transports Ca2+ ions across membranes. When eggs are placed in seawater containing it, the cortical granule reaction is induced. Ca chelators like EGTA prevent the reaction from occurring if injected into a sea urchin egg.

Front 4

Four major events of fertilization.

Fusion of genetic material of sperm and egg -

Back 4

- After the sperm has undergone acrosome reaction and has traveled to the egg, the sperm nucleus separates from the flagellum and mitochondria. In sea urchins, fertilization occurs after the 2nd mitotic division, there is a haploid female pronucleus in the cytoplasm of the egg when the sperm enters.

- In frogs, sperm can enter anywhere on the animal hemisphere. When it does, it changes the cytoplasmic pattern of the egg. Originally the egg is radially symmetrical about the animal-vegetal axis. After sperm entry, the cortical cytoplasm shifts 30° toward point of sperm entry, relative to the inner cytoplasm. A region of the egg that was formerly covered by the dark corticalcytoplasm of the animal hemisphere is exposed by this shift. The gray crescent marks the region where gastrulation is initiated in amphibian embryos.

Front 5

Four major events of fertilization.

Activation of egg metabolism to start development -

Back 5

Fusion of the sperm and egg activates 2 polyspermy blocks. The activation of all eggs appears to depend on an increase in the concentration of free Ca2+. It is also responsible for the re-entry of the egg into the cell cycle and reactivation normally fertilized egg.

Calcium release activates a series of metabolic reactions. Activation of enzyme NAD+ Kinase converts NAD+ to NADP+ (important for lipid biosynthesis - cell membrane production).

Late Responses: activation of DNA synthesis and protein synthesis. The rise in intracellular pH begins with a second influx of sodium ions. It is thought that the pH increase and Ca2+ elevation act to stimulate new protein synthesis and DNA synthesis. Ca2+ inactivates the enzyme MAP Kinase which removes an inhibition on DNA synthesis. The wave of free Ca2+ inactivates MAP Kinase, and DNA synthesis resumes.

Protein synthesis utilizes mRNA's already present in the oocyte cytoplasm. These mRNA's encode proteins such as histones, tubulins, actins and morphogenic factors that are utilized during early development.

In sea urchins, an inhibitor binds translation initiation factors eIF4E and prevents translation from occurring. Upon fertilization, the 4E-binding protein degrades allowing translation of stored sea urchin mRNA's of egg protein synthesis. If the calcium-chelating chemical EGTA is injected, there is no cortical granule reaction, no change in membrane resting potential, and no reinitiation of cell division. Eggs can be activated artificially in absence of sperm by procedures that release free calcium into oocyte.

Front 6

Sea Urchin

Radial holoblastic cleavage -

Back 6

The 1st and 2nd cleavages are meridional and perpendicular to each other. The 3rd cleavage is equatorial, separating the Animal and Vegetal hemispheres. In the 4th cleavage, the 4 cells of the Animal hemisphere meridionally divide into 8 equal blastomeres called mesomeres. The 4 cells of the vegetal hemisphere divide unequally via equatorial cleavage to produce 4 large macromeres and 4 small micromeres. Cells in each hemisphere continue to divide and after the 7th division, the embryo is a 128-cell blastula.

-- The micromeres are able to produce a signal that tells the cells adjacent to them to become endoderm and induces them to invaginate into the embryo.
Ex: When an isolated animal hemisphere is combined with isolated micromeres, a pluteus larva is formed with all the endoderm derived from the animal hemisphere.

Front 7

β-Catenin and micromere specification
(Maternal effect gene product)

Back 7

Responsible for the specification of the micromeres and their ability to induce neighboring cells.
- β-catenin accumulates in the nuclei of cells fated to become endoderm and mesoderm.
- This accumulation appears to be responsible for specifying the vegetal half of the embryo.

Ex: β-catenin is stained by a fluorescent labeled antibody. During normal development, β-catenin accumulates predominantly in the micromeres. When lithium chloride permits β-catenin to accumulate in the nuclei of all blastula cells, the animal cells become specified as endoderm and mesoderm. When it is prevented from entering the nuclei, the vegetal cell fates aren't specified and the entire embryo develops as ciliated ectodermal ball.

Front 8

Sea Urchin

Axis Specification -

Back 8

In the blastula, cell fates line up along the A-V axis established in the egg prior to fertilization.
- The A-V axis structures the anterior-posterior axis, the vegetal sequesters maternal components for posterior development.

The oral-aboral axis, which approximates the ventral-dorsal axis of the embryo, is delineated by the first cleavage plane.
- Lineage tracer dye injected into one blastomere at the 2 cell stage demonstrated that the oral pole of the future oral-aboral axis lay 45 degrees clockwise from the 1st cleavage plane as viewed from the animal pole.

Front 9

Slimy little snails

Holoblastic spiral cleavage -

Back 9

The cleavage planes are at oblique angles forming a spiral arrangement of daughter cells. The first 2 cleavages produce 4 large macromeres. In each successive cleavage, each macromere buds off a micromere at its animal pole. The orientation of the cleavage plane is controlled by maternal effect gene products in the oocyte. The orientation of the spindle at the 2nd cleavage determines the cleavage plane and orientation.

Ex: Injecting cytoplasm from a dextral snail into the egg of a sinister snail results in embryos that are dextral. The reverse is not true suggesting that dextral snails have that dextral snail mothers were placing a factor into their eggs that was absent or defective in the sinister mothers.

Front 10

Slimy little snails

Axis formation -

Back 10

Polar lobe buds off before 1st cleavage and is absorbed by CD blastomeres but buds again before 2nd cleavage and is absorbed by the D blastomeres. The polar lobe contains endodermal and mesodermal determinants and gives D its endoderm forming capacity.

Ex: In trocophore larva, if the polar lobe is removed the remaining cells will divide normally. The result is incomplete larva lacking endoderm, mesoderm organs, as well as some ectodermal organs.

Ex: Specifcy dorsal-ventral axis. When polar lobe material is forced into AB blastomeres as well as the CD, twin larvae are formed, joined at the ventral surfaces.

Ex: The determinants are in the cytoskeleton of the polar lobe. Injecting cytoplasm from polar lobe into blastomeres B did not produce duplicated structures.

Front 11

Spemann & Mangold

Back 11

- Spemann & Mangold used differently pigmented embryos from two newt species.
Dark = Triturus taeniatus
Light = Triturus cristatus
- The dorsal lip of T. taeniatus was transplanted into a gastrula of T. cristatus to become ventral epidermis.
- The dorsal lip invaginated and disappeared beneath vegetal cells.
- The pigmented donor continued to differentiate into the chordamesoderm (notochord).
- As the donor mesodermal cells moved forward, the host began to participate in the production of new embryo, becoming organs that would have never developed normally.
- In this new embryo, the pigmented donor tissue could be seen in the somite along with the host tissue.
- The donor dorsal lip cells were able to interact with host tissue to form neural plate from host mesoderm.
- Eventually a second embryo formed, face to face with the host.
- The ability to form the neural tube and dorsal axis from dorsal lip cells is called PRIMARY EMBRYONIC INDUCTION.

Front 12

1 Maternal and 3 or 4 zygotic gene products

early amphibian development

Back 12

β-catenin: induces dorsal axis formation (maternal)
Ex - β-catenin injected into the lateral side of the embryo a 2° dorsal lip formation will occur.

Noggin and Chordin are zygotic gene products that both inhibit BMP4 & 2. This enables neural tissue to form as needed.

Cerberus is a zygotic gene product. It is responsible for eyes and olfactory placode development. If Cerberus is injected into the posterior region of the embryo, ectopic head formation will occur.

Dickhoff - with Wnt, it induces anterior head formation.
Ex - If an antisense mRNA for Dickhoff is introduced, there's no forebrain formation.

Front 13

Slow block step process -

Back 13

Sperm fusion --> G protein activation --> src activation --> PLC activation --> PIP2 to IP3 --> Ca2+ release --> corticle granule exocytosis --> slow block

Front 14

Explain the origin AND migration of neuroblasts in any part of the bertebrate CENTRAL nervous system.

Back 14

origin - neuroblasts originate in the neural tube formation of the neural ectoderm
Evidence: radioactive thymidine injected into neural tube of rhesus monkey. Autoradiography of brain (neural) tissue is point of interest - neuroblasts can be identified and traced, 100% of radioactive thymidine is incorporated into DNA as long as DNA synthesis is taking place. Thymidine radioactively labeled is incorporated in the DNA of the neuroblast when it is in the lumen, but it migrates throughout the CNS.

migration - glial cells and astrotactin adhesion molecules.
Evidence: an antibody to astrotactin, glial cells cannot bind to the astrotactin and the glial cells migrate out of the CNS.

Front 15

Birds & Mammals

Cleavage -

Gastrulation -

Back 15

Cleavage - Birds have discoidal meroblastic cleavage. Cleavage occurs only in the blastodisc. The first cleavage appears centrally in the blastoderm and others follow to create a single layer blastoderm. The cells lining the yolk don't have complete cleavage, leaving a layer with many nuclei contained in cells that aren't divided from each other (syncytial). The subgerminal cavity is created when the blastoderm cells absorb water from the albumen and secrete the fluid between themselves and the yolk. The area pellucida is created when the deep cells of the blastoderm are shed and die. The area pellucida forms most of the embryo. The peripheral ring of blastoderm cells is the area opaca and the marginal cells are between them. The marginal cells are important in determining cell fate during early chick development.

Gastrulation - Most cells of the area pellucida form the epiblast, while some migrate to form the primary hypoblast. A sheet of cells from the posterior margin of the blastoderm pushes the primary hypoblast cells anteriorly, forming secondary hypoblast (or endoblast). Epiblast forms the embryo while hypoblast forms external membranes. The 3 germ layers are formed from epiblast.

Front 16

Determination of the Anterior-Posterior Axis (vertebrates) -

Back 16

Expression patterns of Pax 2, 5 and 6. These genes are important for formation of forebrain and hindbrain. If gene knockout is done to Pax 2 and 5, the mesencephelon primordia will be missing and the forebrain will connect directly to the hindbrain. Midbrain and cerebellum are missing.

Front 17

Determination of the Dorso-Ventral Axis (vertebrates) -

Back 17

This is determined by epidermis and the notochord. The notochord uses the paracrine factoer Sonic hedgehog to induce ventral formation. Epidermis uses BMP4 and 7 to induce dorsal formation. Zebra fish with mutant BMP genes lack dorsal neuron formation.