What phase in development to embryos usually look the same?
Beginning of neurulation
Describe fate mapping
Traces cell lineages: following individual cells to see what those cells become
Use of molecular dyes and computer imaging to determine what cells develop into
What is an example of a dye that can be used to fate map?
What is a chimeric embryo?
Embryos made from tissues of more than one genetic source
What is GFP
Green fluorescent protein
Altered gene called a transgene
Give cells a virus so that they fluoresce
Describe homologous structures
Organs whose underlying similarity arises from their being derived from a common ancestral structure
Example wing of bird and human arm
Describe analogous structures
Those whose similarity comes from their performing a similar function rather than their arising from a common ancestor.
Example wing of butterfly and wing of a bird. Did not arise from common ancestral structure
What are the two major periods in development?
Embryonic period (up to 8 weeks in humans)
Fetal period (remaining time in utero)
Where is BPA commonly found
What is a fundamental difference between eukaryotic genes from prokaryotic genes?
Eukaryotic genes are contained within a complex of DNA and protien called Chromatin.
Protein component is around have the weight of chromatin and is composed mostly of histones.
What is the on/off switch for gene expression?
Describe exons and introns
Exons are the regions of DNA that code for protein.
In between exons are intervening sequences called introns that have nothing to do with the AA sequence of the protein.
In the drosophila oocyte what does Bicoid protein initiate?
Head and thorax formation
What are the two things that Bicoid does in Drosophila. Explain
Bicoid can act as a transcription factor
-This activates genes such as hunchback that are necessary for forming the fly anterior
Also acts as a translational inhibitor of those genes such as caudal that are critical for making the fly posterior.
Describe Model Organisms
Can be both animals and plants
An experimental tool to investiage a particular biological process. Model organisms are typically easy to grow in a lab setting and have been an effective means of investigating the cause of human disease without conducting unethical research directly on humans.
Describe how cloning was done in sheep
Take the oocyte from a sheep and remove the meiotic spindle (where the genetic information is)
Transfer cells grown in G1 stage into the enucleated egg
Fuse with electric current, and transfer to a uterus.
What controls cell fate?
If each cell has the same genetic content in embryogenesis, how do different regions evolve into different parts?
While the nucleus controls cell fate, differential gene expression underlies cell fate specification .
Describe the basic steps in protein formation
Transcription of genetic information
Describe how transcription factors form bridges? What is the point of this?
They loop the chromatin such that the transcription factors on enhancers are brought into proximity with the promoter.
The point of this is to link the genetic information together.
Describe the mediator complex
Usually bridges the enhancer and promoter regions.
The mediator initiates a chromatin loop connecting the enhancer and promoter
Describe the Fgf Family
fibroblast growth factor
Often work by activating a set of receptor tyrosine kinases called fibroblast growth factor receptors (FGFRs)
What is essential in enabling the Hedgehog protein to anchor to its receptor cell's cell membrane?
Describe the hedgehog family
Often created by the embryo to induce particular cell types and to create boundaries between tissues
Vertebrates have at least __ homologues of the Drosophila hedge hog gene
___ of the Hedgehog family function by binding to a receptor called ___
Patched protein prevents ___ protein from functioning
When Hedgehog binds to ___ the protein's shape is altered so that it no longer inhibits ___. this releases proteins from microtubles and prevents it from being cleaved. The protein enters the nucleous where it acts as a ___ ___ of the same genes it used to repress.
What is the hedgehog pathway extremely important in? What happens when animals are homozygous with a mutant allele?
Hedgehog pathway is extremely important in vertebrate limb development, neural differentiation and faction morphogenesis.
When mice were made homozygous for a mutant allele of Sonic hedgehog, they had major limb and facial abnormalities. The midline of the face was reduced and a single eye formed in the center of the forehead
Members of the Wnt family usually interact with a pair of transmembrane receptors called ___.
What is a frizzled?
A transmembrane receptor that Wnt binds to
In the absence of Wnt, the transcriptional co factor ___ is constantly being ___
Describe Wnt Signalling (Briefly)
Wnt binds to frizzled
Frizzled interacts with a protein called Disheveled which stablizes the axin and prevent phosphorylation of B-catenin.
B-catenin binds within the nucleus and activates Wnt responsive genes
What is a natural inhibitor of the Wnt pathway
What is Wnt essential in?
Critical in establishing polarity of insect and vertebrate limbs, promoting the proliferation of cells, and in some steps within the mammal urogenital system.
Related to Wingless gene in Drosophilia
What is BMP a member of?
The TGF-B family
What are important families within the TGF-B super family
Nodal and activin
What is distinguishable from the BMP family
They have 7 rather than 9 conserved cysteines
What does BMP stand for
Bone morphogenetic proteins
What is the function of BMP
Regulate cell division and apoptosis
Regulate cell migration and differentiation
What are Nodal and activin proteins important in?
Specifying the different regions of the mesoderm and distinguishing the left and right sides of the vertebrate body axis
The TGF super family activates the ___ Pathway. The TGF __ binds to the TGF ___ which allows for two ___ to bind. The activated ___ can now phosphorylate the ___ Proteins. Eventually the phosphorylated protein forms a ___ which enters the ___
Transcription factor complex
Why is notch unique?
It extends through the cell membrane and it's external factor contacts with Delta of another cell.
How does Notch work?
Its external part of the cell binds to Delta (a ligand from another cell) which causes a Notch to change shape activating a protease. The activation of the protease cleaves notch, which enters the nucleus and binds the transcription factor, which transcribes target genes
What are classical embryological methods of Developmental Biology? (4)
Observation of cells
Cutting up embryos
What are Molecular Biology methods of Developmental biology? (7)
Gene expression analysis
Gene loss of function
Gene gain of function
Describe In situ hybridization
A RNA is made complementary to a specif mRNA and inserted through a hole in the cell membrane.
The RNA is labelled (radiolabelled)
One example of a label is Digoxigenin
The label is then bound using an antibody
After phosphorylation the colorless compound becomes dyed.
What is another name for 'loss of function'
What is a method to analyze gain of function?
Creating a knockout organism
How are knockout organisms created?
Essential create a mutant gene and inject it into the embryo.
Inject heterozygous cells into blastocyst
Inject blastocysts into the uterus
Formation of a chimeric animal.
Then breeder the Chimeric animals to the wild type to get more wild types
Breed wildtypes together to get a homozygous mutant lacking the gene.
What are cadherins
Calcium Dependent adhesion molecules
Critical for establishing and maintaining intercellular connections
Cadherins are anchored inside the cell by a complex of proteins called ___
What are the four stages of cell migration?
Polarization, where a cell defines its front and back
The protrusion of the cell's leading edge. Mechanical force for this is the polymerization of the actin micro filaments at the cell membrane.
The adhesion of the cell to its extracellular substrate. Integrins span the cell membrane and connect the extracellular matrix outside the cell to the actin cytoskeleton on the inside of the cell.
Release of adhesions in the rear, allowing the cell to migrate in the forward direction.
What is DDT
Affects animals at the top of the food chain
In embryos can cause preterm birth and low birth weight
some links with cancer
it is an estrogen receptor binding chemical
How are chromosomes numbered?
Which organism can be used to study gametogenesis?
Why are nemotodes studied?
Can watch cell division for the entire organism (very few cells)
convenient and easy to raise in the lab
Why are chickens losing favor in developmental biology?
Currently unable to make mutations of genes
Much more limited scope of techniques can be done
What is an advantage of zebra fish?
What was the key study that was done to show that the key repository for information was the nucleous?
Take a nucleous from an albino frog and inject that in an unfertilized embryo.
New embryo is albino
How do ligands work?
Bind to a ligand-binding domain that can activate an enzymatic activity
Activates an active protein
How do two cells that are touching each other signal?
What is ISH?
In Situ hybridization
What does the degree of colour in In Situ Hybridization mean?
The proportion of the gene expression
Does not give an exact numerical value
Why are Drosophila used?
Short generation time
Four pairs of chromosomes
Salivary polytene chromosomes are highly duplicated and lined up
first behavioural screens
Describe the early divisions in drosophila and how it is different
Nuclear division - not cell division
It is a syncytium - multiple nuclei
Any kind of timing that occurs in early division is going to be synchronous
What is a key step in alternating the cell cycle?
Activation of the zygotic genome in one of the following transition:
MBT (Mid blastula transition)
MZT (Maternal zygote transition)
Describe the genetic screen
Mutagenize generation ZO (randomly mutates some genes in the organism)
Cross to Wild type organism to generate F1 (One of the chromosomes will be a mutant, One will be normal)
Intercross F1 generation to generate F2 (some chromosomes will be homozygous - both mutant)
Screen progeny (F3) for defects in segmentation
What are GAP genes?
Large portions of embryo are deleted
Gene hypothesized to function in defining segment identity.
What are the different segments in Drosophila?
The first ___ cell cycles in Drosophila are in a syncytium
The creation of the __ phase is dependent on activation of zygotic __
The activation of what drives cells into mitosis?
Activation of Cyclin E
Why does the embryo have different compartments?
What are pair rule genes
Function to specify parasegmenta identity
Affect embryo two-segment periodicity
Describe segment polarity genes
Regulate a subdomain of every segment
Also function to define anterior vs posterior polarity within segments
Specify cell identity within segments
What functions to define anterior vs posterior polarity within segments?
Segment polarity genes!
What is the French Flag Model?
A model to show that different gradients can cause different outputs.
There is a blue, white and a red colour indicating an output and a correlating threshold for each of the colours
What transports bicoid mRNA?
What does bicoid control?
Describe the normal development of an embryo compared with the development of a bicoid deficient mutant
Normal development has bicoid at the anterior end of the embryo.
An embryo will have two tail regions in a bicoid deficient mutant
What was an experiment that was done to determine that bicoid controlled anterior fate?
Bicoid was added to the anterior end of a mutant => lead to normal development
Bicoid was added to the middle of the mutant => Formation of a head in the middle of the ebmryo, with two tail regions either side
Bicoid was added to the posterior region of the embryo => two heads with a tail in the middle.
What does nanos do?
Nanos is on the posterior side of the embryo
When in Drosophila development do cell membranes form?
Not until after the 13th nuclear division
In Drosophila, what forms the dorsal-ventral polarity in the embryo?
Gurken protein enters the nucleus and ventralizes the cell
What is the function of Dorsal? Where is it located?
Dorsal is located all throughout the embryo.
It is only transcribed into the nuclei on the ventral region of the embryo.
This represses the dorsal cell types, causing for the ventralization of the region.
Yes, Dorsal leads to Ventralization. Cray.
How many segments are affected in a pair rule gene mutant in drosophila? Segment polarity?
Does protein always show the same distribution of mRNA? What does this mean?
No, because the step from RNA to protein is regulated
Can create a gradient
What does DNA make?
RNA, which makes proteins
Where is hunchback mRNA made? What does it do?
Hunchback protein is made in the anterior region of the embryo in drosophila. This allows for the the creation of Hunchback protein and promotes anterior structures.
How does nanos interact with Hunchback?
Nanos in the posterior region of the embryo inhibits hunchback protein and allows for abdominal formation.
Why are transgenic flies made?
To examine regulation of pair rule genes
Where is the caudal protein in drosophila made?
The Posterior region
Bicoid is made in the ___ region and Nanos is made in the ___ region (Posterior or Anterior_
What is found in between segment polarity genes?
Describe the wingless molecule
A small polypeptide is made by the cells in the embryo
Diffuses out of the cell
Diffuses in a gradient
There are lots of cells along the embryo that releases the cell, so there are many small gradients of Wg expression
The polypeptide binds to a receptor in adjacent cells
How do wingless and engrailed work together?
The cell that creates wingless is adjacent to the cell that creates engrailed
Engrailed has a receptor that can detect the wingless gene
Releases a hedgehog signal that goes to the initial cell and binds to patched. This causes for more wingless expression - signal amplification.
Essentially the two are required for normal development. Absence of one causes the mutation
__ and __ are inhibited by __ inhibiting dorsal structures of the embryo. This means that this section of the embryo will develop in to the __ region
How do we make the termini of the embryo in drosophila
Interact with support cells containing a ligand and a receptor. Eventually transcription factors are activated. Torsolike activates torso at the poles of the embryo, causing for the formation of the poles.
Bicoid determines head vs. tail
What is homeosis? Provide an example
The transformation of one body part/segment into another via the activation, mis expression or inactivation of a critically important developmental regulator
An example would be Ultrabithorax: fly with two sets of wings
What is ultrabithorax? how is it formed
Fly with extra set of wings
Example of homeosis
Loss of Ubx gene function
What is Antennapedia? How is it formed?
When a fly has legs growing on its head where antenna should be growin
An example of homeosis
Caused by gain of Antp gene function
What is the function of Hox transcription factors?
DNA binding domain
Where are genes causing homeotic mutations located in drosophila?
All on the same chromosome next to each other
What is meant by Hox genes are highly conserved?
Genes are expressed in different animals, just in varying clusters. Less in flies, more in mouse etc.
Some sections are elongated within the Hox, and other Hox sections are removed.
What is a Hox gene?
A homeotic selector Gene
Describe fertilization in sea urchin
Sperm contacts jelly layer
Digestion of jelly layer
Binding to vitelline envellope
Fusion of acrosomal process membrane and egg membrane
What is the final stage in sperm development? Where does it occur?
Occurs in female reproductive tract
How are sperm usually attracted to the egg?
In echinoderms, how are sperm provided with direction to the egg? Why is this needed
This is needed because of the external fertilization process
Sperm-activated peptides are released along with the eggs.
What is important in the acrosomal reaction (in sea urchins) in mediating recognition
A protein called bindin.
Describe the acrosomal reaction in sea urchins
Bindin binds to the surface of the egg
Actin microfilaments extends by actin polymerization.
What facilitates species specificity in acrosomal reaction?
How is polyspermy prevented (sea urchins)
Fast block to polyspermy: Na Channel-dependent depolarization of membranes
Cortical granule reaction (Slow block to polyspermy): Cortical granules fuse with the egg cell membrane => form a fertilization membrane.
Describe early cleavage divisions in the frog
Mesolesical egg - more yolk at ventral pole
Displaced radial cleavage
Cells at the bottom larger
Where are the animal pole and vegetal poles located?
Vegetal pole is at the bottom => all of the yolk to support the embryo at the animal pole (top)
Describe cell movements of the frog embryo
Blastocoel originally at the top of the embryo
The yolk at the bottom moves up, displacing the blastocoel and creating the archenteron (another area of space)
Then the cells being to fold along the inside of the sphere giving rise to the mesoderm
What does gastrulation give rise to?
Three tissue layers
In frogs, how is the archentron formed?
Bottle cells change shape dramatically
Main body of each cell is displaced toward the inside of the embryo while maintaining contact with the outside surface.
What is another name for archentron
Describe early zebra fish development
telolecithal - lots of yolk throughout (incomplete meroblastic cleavage)
Formation of a blastodisc - a kind of bubble that forms out of the embryo
Bottom part does not divide (mostly yolk)
Formation of Yolk syncytial layer: yolk all in one area
Continued separation. As yolk continues to push up, cells continue to be pushed along the side (like headphones).
What is the shield?
The shield is near the animal pole in zebra fish, and is on the side of the egg that becomes the dorsal region of the egg.
Mesoderm and dorsal inducing signals are released
*Note* In frogs dorsal creates the dorsal region. In flies everything is fucked the hell up.
During gastrulation in zebra fish, describe the movement of cells
Cells involute and ingress (fold inwards)
Move toward the midline of the embryo/egg
What is significant about the embryonic shield?
Cells of the epiblast and hypoblast intercalate to form a thickening.
The cells converge and extend anteriorly. (move up)
What parts of the blastula give rise to the ectoderm, mesoderm and endoderm?
Animal cap cells => ectoderm
Marginal cells => Mesoderm
Vegetal cells => endoderm
The animal is usually converted into the __ but can be converted into the __. How?
This is done by factors released from vegetal cells - usually when the marginal cells have been removed experimentally
What are key modulators of development?
What do cell lines use for continued proliferation?
What is the Nieuwkoop center?
The dorsalmost vegetal cells in frogs that are capable of inducing the organizer.
What is the 'organizer' in xenapus?
dorsal lip cells and their derivatives (notochord and head endomesoderm)
They induce hosts ventral tisues to change fates to form a neural tube and dorsal mesodermal tissue
Organized host and donor tissues into a secondary embryo with clear anterior-posterior and dorsal-ventral axis
Describe a transplantation experiment
The experiment showed that only one of the tissues in the early gastrula has its fate predetermined
This tissue was dorsal lip of the blastopore (Later named the organizer)
When the tissue was transplanted into the presumptive belly skin region of another gastrula it continued to be dorsal blastopore lip but also initiated gastrulation and embryogenesis in the surrounding tissue.
What are XTC cells?
Cells that are grown in a dish
They grow without adding a growth factor
This suggests that they secrete their own mesoderm inducing signal
What is the importance of XTC cells?
XTC cells create growth factor - this was important in diagnosing what the specific mesoderm inducing signal is.
How was actin determined to be the mesoderm inducing factor?
through cell culture work. Both XTC and Actin A have mesoderm inducing factor
However Actin is not the mesoderm inducing factor.
Molecular screening was tried to find out what __ was. How was this done?
Mesoderm inducing factor
Separated the eight cell embryo into four vegetal poll cells and four animal blastomeres
They isolate the mRNA's from the two types of cells and compare
Convert mRNA's into a label probe using a radioactive tracer and then hybridize and compare.
What is VegT?
A transcription factor that is important in activating the mesoderm inducing factor
Regulated nodal related
Nodal related is the mesoderm inducing factor
What is the mesoderm inducing factor
Xnr in frogs (Xenapus nodal related)
What did the study of the frog embryo give us?
Allowed us to find the mesoderm inducing factor (nodal related)
Also large number of embryos - a very large amount of source material.
What does VegT regulate?
Derriere and Nodal related genes
Induces them so that they can form mesoderm
What is the source of mesoderm induction in zebra fish?
Yolk syncytial layer
What is the most common human forebrain birth defect?
What does Cyclopamine cause?
Cyclopia in sheep
What induces the ventral forebrain?
What is the prechordal plate?
The embryonic shield forms the prechordal plate
The prechordal plate cells are the first to involute and migrate toward the animal pole
Responsible for inducing ectoderm to become neural ectoderm.
What are some downfalls in using the frog embryo
Takes a long time to reach maturity
What is a genetic screen
Very powerful methods in an unbiased method to ask the embryo what genes are important for specific functions/characteristics
What are a couple forebrain zebra fish mutants? What is the gene of importance
One eye pin head
They lack mesoderm
Gene of importance is squint
Where is the squint gene found? In what Organism? What happens if it is removed? Added?
Found in the group of nuclei between the yolk syncytial layer and the embryo
In zebra fish
Remove mesodermal induction
Increased mesoderm induction
What is a morphogen? How can we help conceptualize this?
A molecule that:
Acts directly on cells at a distance
Usually present in a gradient
causes fate changes in dose-dependant manner
The French flag model can hep conceptualize this.
What is the definition of an organizer?
A group of cells that can alter the fates of surrounding cells.
Importantly, once formed, an organizer independent of outside signals. This means that you can transplant an organizer to another place and it will retain its activity.
When does Dorsal-Ventral specification start in Xenapus?
Starts as early as the 2-4 cell stage.
What is required for dorsal specification in Xenapus?
How was the source of dorsal identity found?
Through transplantation experiments
Identifying the Nieuwkoop center and dorsal organizer
Look for localized proteins and mRNA that move during cortical rotation
What is the gene in Xenapus that ventralizes the embryo?
Where on the Xenapus embryo is B-catenin stabilized?
The dorsal side of the embryo
After fertilization in a xenapus, what happens? In terms of genes
There is cortical rotation
The Dishevelled protein (Dsh) and Wnt 11 mRNA relocalize to the presumptive Dorsal side of the embryo
This leads to the dorsal enrichment of B-catenin
What does B-catenin regulate (in xenapus)
What drives Nodal? What drives it in Xenapus? What happens when Nodal drives?
Nodal is the mesoderm inducing factor
B-Catenin + vg1 + Veg T drives Nodal
Low Nodal related leads to ventral mesoderm, Nodal related high leads to the organizer
Organizer specifies the dorsal region
What are the organizer molecules? What do they do
Chordin, Noggin, Follistatin and Xnr 3
Specifies the dorsal region of the embryo
What is the dorsal organizer in zebrafish?
The zebrafish shield
What does the removal of ndr1 containing cells result in, in zebrafish?
The loss of dorsal structures
Localization of Nodal apparent in the zebrafish?
The 4-cell stage
What is swirl? What does the overexpression of swirl cause?
It is a BMP mutant phenotype
over expression of swirl causes ventralization
Describe the overall D/V patterning in zebrafish
Dorsal region: Wnt binds to Dsh which leads to beta-catenin allowing for organizer to be expressed. This causes for dorsalization. The organizer releases three molecules that prevent the ventral region from developing (chordin, noggin and follistatin)
Ventral region: Gdf6 and BMP2/7 are expressed and lead to the ventralization of the section.
What are the three primary vesicles of the brain?
Forebrain: Telencephalon and Diencephalon
Hindbrain: Metencephalon and Myelencephalon
What is Frzb?
Wnt binds to it instead of the receptor (Frizzle)
When there is a lot of Frzb, there is no Wnt binding
What are Row 1 cells?
The cells that make the anterior neural border
What does the injection of a synthetic Wnt in an embryo do?
No development of anterior markers in neural tube development
What happens when synthetic Frzb is injected into an embryo?
No development of posterior markers
Lots of anterior neural tube development
What does Frzb and Wnt tell us?
Frzb is expressed in the anterior region of the neural tube
Wnt is expressed in the posterior region of the neural tube
What sort of pattern are Hox genomes expressed in?
What is a Rhombomere?
A hindbrain segment of an embryo
Hox genes show exquisite segmental expression pattern in the hindbrain
What is a morpholino
A molecule that contains a multiple nucleotide analogue
Derivative of oligonucleotide
What is the transgenic approach in how Hox genes are regulated?
Find candidate regulatory sequences
Create a DNA construct with candidate regulatory sequences linked to a reporter
Integrate into organism
Does reporter recapitulate expression pattern?
Analyze your newly discovered regulatory sequence
Test putative regulators
What is retinoic acid a derivative of?
Why would one want to make a transgene?
To label a portion of the embryo
To study gene regulation
To overexpress/ectopically express gene (ectopically = different region)
What does conservation in genes mean?
If genes mutate and they are kept it is likely that they serve a purpose. There is some genetic noise, but it can be used to find candidate regulatory sequences
Regulatory sequences being conserved area between species
What is unique about the dorsal ventral axis in flies?
It is flipped in comparison with other animals
Describe the retinoic acid signaling pathway
Aldh1a leads to retinoic acid signalling
leads to retinoic acid receptors (RAR) and Retinoid receptors (RXR)
These receptors lead the expression of the Hox gene
cyp26 inhibits retinoic acid (creates an enzyme that degrades RA)
What are the expression patterns of cyp26 and aldh1a?