Download Outline 2 Part 2

BIO 185: Developmental Biology – Fall 2003
Outline 2 – Part 2
II. Early Development: Wow, so we are going to talk about embryos after all! The penetration
of the egg by a sperm cell and its subsequent resistance to all others is very interesting but what
happens shortly afterward is why we’re here. Once the egg begins to respond to the presence of the
sperm development of the organism begins. The events that lead to the first asymmetric division go
far beyond the mixing of genes. Many events are unrelated to the pro-nuclei. Not surprisingly, these
ancient events are similar in different organisms. Real differences between developmental strategies
begin during the formation and development of blastula and gastrula stage embryos. This section
discusses and compares these early events in several species. Finally, we will look at the molecular
mechanisms by which embryos establish their ups and downs and rights and lefts, aka “Axis
Formation”. This is where it all begins!
C. Assymetric Division of the Ovum: We have talked about this process in bits and pieces
during these first few weeks of class. What do actually know about this process?
1. The Activation of Egg Metabolism (p. 203)
a. The egg is divided into different domains to begin with.
1. The animal/vegetal axis and the animal vs. vegetal hemispheres (and poles).
2. The cortical vs. subcortical cytoplasms.
b. Calcium waves increase cytosolic [Ca++] ten-fold in nearly all species.
1. Chelating Ca++ stops development, adding it without sperm can fire up early events
2. The successive waves produce successive events.
a. Early Responses
1. Activation of membrane biosynthesis by activation of NAD kinase
2. Restoration of cell cycle through inactivation of cyclin, etc.
b. Late Responses
1. Stimulation of protein synthesis through interaction with high pH
a. Protein made from stored egg mRNA, not sperm mRNA.
2. Stimulation of DNA replication by inactivation of MAP kinase
3. Movement of cytosolic components.
c. Later events require a given number of waves.
1. Maybe concentration, maybe number of waves.
c. Degradation of sperm mitochondria and their DNA occurs in the egg.
1. This DNA is protected from recombination mutation, isn’t diluted with male DNA
2. That’s why we track historical species and migrations through mito DNA.
3. Rearrangement of Egg Cytoplasm (p. 210)
1. Not dramatically visible in all organisms, but you can use dyes to show that it occurs.
2. The sperm can penetrate at any point around the circumference of the sphere.
1. Entry causes rotation of the cortical cytoplasm 30o toward entry point.
2. The rotation is caused by microtubule arrays connecting the cytoplasms.
a. Interestingly, the arrays are composed of tubulin from both sperm and egg.
b. The centriole (initiation site) guiding them is from the sperm.
4. The First Cell Division Follows Rearrangement.
1. Calcium waves have started cell cycle activities.
2. Cytosolic rearrangements have set up the essential asymmetry.
3. The cleavage plane forms at the site of pronuclear fusion.
D. Cleavage and Blastula Formation: In most organisms, cleavage starts as a highly
accelerated mitosis that divides the cytosol of the egg into many small cells based on the stored
instructions in the maternal mRNA. The developing cells then undergo a transition to expression of
new mRNA’s from the newly combined genome and differential development in the organism is
truly off and running. The blastula is the structurally organized cell mass that forms by these cell
divisions and by fluid secretion. It is this structure that prepares the embryo for the first migratory
restructuring that comes with gastrulation. Most animals are similar in their basic mechanisms of
development at this stage and we’ll talk about several, using the bird as our primary model. We will
then talk about a group of animals that have developed some truly new strategies for these early
stages - the mammals.
1. The Basic Mechanisms of Cleavage (p. 222)
a. Rapid Cell Divisions Lacking the Gap Phases of Mitosis Divide Up the Egg Cytosol
1. G1 and G2 are the times that cells grow in size prior to division
2. Cells only have time for S (synthesis) and M (mitosis) phases
3. The rate of nuclear multiplication is off the chart
a. Frog egg can divide into 37,000 blastomeres in 43 hours.
b. Drosophila forms 50,000 cells in 12 hours – 10 minute cell divisions!
4. Most organisms have extremely synchronous cleavages and exponential growth.
b. Cycling of the Mitosis-Promoting Factor Through Expression and Degradation of Cyclin B
1. MPF Activity is High in M-phase and low in S-phase
2. MPF has two subunits – Cyclin B and Cyclin-Depenedent Kinase (cdc2)
3. Cyclin B is translated from maternal mRNA, then the protein is degraded
4. When Cyclin B is Attached, cdc2 Phosphorylates its Target Proteins
a. Phosphorylation of histones causes chromosome condensation
b. Phosphorylation of lamin causes breakdown of the nuclear envelope
c. Phosphorylation of myosin causes organization of the mitotic spindle
c. The Mid-Blastula Transition to Genomic mRNA Direction
1. Eventually the Stored Mitotic Regulators Run Out
2. Synthesis of mRNA from Combined DNA is Now Driven by Blastomere Position.
3. G1 and G2 are reintroduced and cells start to grow during mitosis.
a. Frogs reintroduce them both after12 cleavages
b. Drosophila reintroduces G2 after #14, G1 after #17
4. Synchronicity of Cell Divisions is Lost as Cells Make Different MPF Regulators.
d. Patterns of Embryonic Cleavage
1. Pattern Depends Primarily on the Amount and Distribution of Yolk in the Egg
a. The vegetal pole is defined as yolk-rich, the animal pole as yolk-poor
b. A ton of yolk makes the cleavage occur in a disk shape at the animal pole.
2. Different Inherited Patterns of Cell Division Can Also Affect the Cleavage Pattern.
a. Most obvious in yolk-poor eggs.
d. Specification of Cell Fates During Cleavage
1. Due to Assymetric Distribution of Patterning Molecules During Cleavage
a. Passive acquisition due to location of cytoskeletal attachment
b. Active transport to predetermined cells
c. Organized association with a single centriole.
2. The Chickens – Now Representing Birds, Fish and Reptiles (p. 354)
a. Meroblastic Discoidal Cleavage
1. Initial cell divisions are all meridional and highly synchronous.
2. All “cells” start out continuous with the yolk at their base.
b. Formation and Development of the Blastoderm
1. Equatorial (vertical) divisions then form multiple layers of cells.
2. Most cells lose their continuity with the yolk by physical separation.
c. Dissociation of the Blastoderm from the yolk
1. Cells of the blastoderm absorb water from the albumin and secrete it again.
d. Formation of the Two-Layered Blastula
1. The epiblast contains all of the embryo cell fates
a. Apoptosis in the inner cells of the blastoderm leave a single layer.
b. A ring of multiple layers is maintained at the edge – the marginal zone.
2. The hypoblast contains most of the non-embryo cell fates
a. Formed by a combination of marginal zone cells and delaminating epiblast
3. The Mice – Now Representing all of Mammalia (p. 363)
a. Holoblastic Rotational Cleavage
1. Like most things mammalian, these are the last things we learn about.
a. Very difficult to study.
b. The human egg is less than 1/1000 the volume of xenopus eggs
1. 100 m in diameter – barely visible to the human eye
c. Produced in much smaller numbers and far less accessible.
d. Egg is encased in the zona pellucida (extracellular matrix “shell”).
1. Keeps it from implanting in the fallopian tubes.
2. Extremely slow cleavage – probably related to the delayed traverse down the tubes
a. 12-24 hours apart
b. Start in the oviduct
b. Very different cleavage pattern.
1. First cleavage is normal meridional.
2. Second is half meridional and half equatorial.
3. Cleavages are asynchronous.
a. Often contain odd numbers of cells during development.
4. Cells start out loose and then undergo “compaction”
a. Express tight junctions and gap junctions.
c. Mammalian combined genome is active much earlier than most organisms.
1. The switch from maternal mRNA’s occurs at two-cell stage in mice and goats!
d. Formation of the morula at about the 16-cell stage.
1. Cells become either inner cell mass or trophoblast.
a. Inner cell mass cells have all embryo fates.
b. Trophoblasts have non-embryo placental fates.
c. Can divide into one or the other until 64-cell stage, then are committed.
2. First full-blown differentiation in the mammalian embryo.
e. Cavitation and Blastocyst Formation.
1. Trophoblasts pump sodium in to the morula central cavity, water follows
2. The water filled cavity is called a blastocoel and the embryo a blastocyst
f. Escape from the Zona Pellucida and Implantaion in the Uterus.
1. The blastocyst secretes proteases and cuts a whole it can squeeze through
2. The blastocyst then cuts a whole and settles into the lining of the uterus
E. Gastrulation: Gastrulation is the highly ordered and integrated movement of the cells of the
blastula to form the three primordial layers: the endoderm, mesoderm and ectoderm. This is one of
the most amazing events in embryology. Nearly all organisms undergo the same basic processes,
although they look very different due to the yolk-determined shape of the blastula. The formation of
the blastula established the starting positions of a great number of cells – gastrulation changes
everything!
1. Formation of the primordial layers.
a. The blastula, either inner cell mass or epiblast, carries all the potential cell fates
b. The endodermal progenitor cells must find their way deep into the center of the embryo.
c. The mesodermal cells must migrate less far and form an intermediate sheet.
2. A few key concepts and terms
a. Epithelial in the embryo denotes a sheet of any kind.
b. Epiboly is the word for migration of a sheet of epithelial cells.
c. Mesenchymal denotes individually migrating cells.
d. Delamination is what epithelial cells do when they start to migrate as mesenchymal cells.
3. Gastrulation in the Yolk-Rich Chick Embryo (p. 356)
a. Starts from the two-layered blastula: epiblast and hypoblast with blastocoel between
1. Remember: the epiblast carries all embryonic cell fates.
b. Formation of the primitive streak.
1. Arises as a lump in the posterior marginal zone of the epiblast
2. Cells become migratory and digest the matrix beneath them.
3. They close in tight and begin to migrate anteriorly – appear to be one population.
4. The front remains thickened and is called Hensen’s node.
5. The migration is coordinated with the extension of the secondary hypoblast.
6. Streak extends to ~75% of area pellucida.
c. The primitive groove.
1. Anterior migration leaves a digested depression in its wake.
2. Allows epiblast cells to migrate through into blastocoel
d. The initial migration through the groove.
1. Mesenchymal migration through the groove then moves anterior.
2. Form the endodermal and mesodermal structures of the head and neck.
3. Presumptive endoderm displaces the hypoblast as it moves.
e. The second wave of migration forms two deep layers throughout the embryo.
1. Mesenchymal cells continue to pour over the lip of the groove.
2. The deepest migrators become endoderm – finished in about 14-18 hours.
3. Many times their number form the mesodermal layer and this takes much longer.
f. Regression of the node leaves the notochord.
1. The node then regresses back toward posterior.
2. At this point all cells left on surface have ectodermal fates.
3. Notochord forms in the poterior direction in wake of regressing node.
4. All development of the embryo is head to tail due to this pattern.
g. Epiboly of the ectoderm surrounds the entire yolk.
1. Herculean task – takes four days!
4. A Couple of Alternative Designs
a. Amphibians are yolk-intermediate and more spherical.
1. Use a blastopore rather than a primitive streak.
2. Most migrations are epiboly.
b. Mammals are yolk-poor and spherical.
1. Follow the exact pattern of the discoidal chick.
F. Axis Specification: There are three principle axes in the body: anterior-posterior, dorsalventral, and right-left. How do the cells of the embryo know where to go to become head vs. tail?
How does the heart know to form on the left side of the body? A lot of inquiring minds want to
know- and slowly we are figuring it all out.
1. Axis Formation in the Chick (p. 360)
a. Specification occurs during cleavage, formation occurs during gastrulation.
b. Dorsal-Ventral Axis Determination
1. Primarily pH determined
a. pH of albumin “above” epiblast is high (9.5)
b. pH of blastocoel “below” epiblast is low (6.5)
2. Gives a transmembrane potential of ~25mV
3. The axis can be reversed experimentally by pH or voltage changes
c. Anterior-Posterior
1. Primarily gravity determined
a. Egg is rotated in the shell gland for 20 hours at 10-12 revolutions per hour
b. Lighter yolk components accumulate at one end
c. Tips up that end of embryo and makes it posterior
1. No one’s 100% certain how but there are some theories out there.
2. One is that this gives a starting point for 2o hypoblast growth
3. Displacement of 1o hypoblast changes the growth factors made.
2. Formation of the “organizer”
a. It is known that this is where the “organizer” will form.
1. All of the marginal zone is capable, but only posterior is site.
b. Cells in the posterior marginal zone start to express VG1 and nodal
1. Forms an active group of cells called the Nieuwkoop Center.
c. The Nieuwkoop Center signals other cells to form Hensen’s Node
1. This starts the primitive streak.
d. Henson’s Node then becomes the mobile “organizer”
1. First it expresses FGF8, which preps ectoderm to become neural
2. Then expresses sonic hedgehog, chordin, noggin and nodal
3. Organizes gastrulation on the way out
4. Organizes notochord formation on the way back
c. Left-Right
1. Growth factor and transcription factor determined
2. At maximum extension of primitive streak:
a. Activin expression begins on right side of embryo
1. No doubt something Henson’s Node did.
2. Stops sonic hedgehog expression on that side only
3. Stimulates FGF8 expression on that side only
b. Downstream of a complex cascade two transcription factors differ.
1. snail is expressed on the right side
2. Pitx2 is expressed on the leftside
3. Experimentally reversing any step in the cascade randomizes left-right axis.
a. The heart and spleen have a 50:50 chance of being on the right side
b. The liver has a 50:50 chance of being on the left side
2. Axis Formation in Mammals (p. 375)
a. The dorsal-ventral and left-right axis are still kind of a black box.
1. Dorsal-ventral appears related to contact with trophoblasts vs. blastocoel
2. Left-right appears related to differential transcription factor expression
a. Perhaps similar to chick – situs inversus gene
b. Anterior-posterior patterning
1. There appear to be two “organizers”
a. One at each end producing double gradients of secreted molecules
2. Ultimately leads to hox gene expression and regional patterning