Zool 303

^{A single celled organism called a zygote}

• Ectoderm (outer layer)
• Mesoderm (middle layer)
• Endoderm (Internal layer)

• Ectoderm: Outer surface, Central nervous system, neural crest
• Mesoderm: Notochord, Bone tissue, Red blood cell
• Endoderm: Digestive tube, stomach cell

• Chemicals that alter embryonic development
• Ethanol - growth and mental retardation
• Thalidomide
• Estrogen receptor binding chemicals
• DDT
• Polychlorinated biphenyls (PCB)
• TCDD (Tetrachloro dibenzodioxin)
• BPA

• A teratogen
• Developed as sedative and anti-emetic. Not toxic in mice or healthy humans
• Causes malformations in babies

• Trisomies and Monosomies
• Copy number variations
• Point Mutations

1 in 100

• Fertility.
• Humans have a fertility of around 20%, where as close ape species have a fertility of around 70%

Usually during anaphase two pairs of homologous chromosomes are paired. In trisomies they are not paired equally and the division in metaphase 2 is unequal.

• Discovered in 1862
• Extra copy of chromosome 21
• Impaired cognitive ability ,distinctive craniofacial features and decreased stature

• Chromosome 13
• This is because the chromosomes are organized by size, and amount of genetic information.

• 1 in 12 000 live births
• Rarely survive 1 year
• Trisomy 13

• 1 in 6000 live birthds
• Median lifespan 10 days
• Trisomy 18

• Klinefelters Syndrome: 47 XXY
• Turner Syndrome 45 XO

• Deletion: Removal of one section of genetic information
• Duplication: Copy of one area of genetic information. Twice that amount
• Inversion: The order of genetic information for an area of the chromosome is flipped
• Insertion: Insertion of extra information into a chromosome
• Translocation: Trading of information from one chromosome into another and vice-versa

• Array Comparative genome hybridization
• DNA from a control individual and patients are individually labeled with fluorescent dye then compared.

Point blank mutations

Changes to the nucleotide sequence of the genetic material of an organism

• Germ line mutation: Hereditable
• Somatic: Somatic cell changes

• Gain-of-function = increase in protein activity
• Loss-of-function = Decrease in protein activity

• Dominant or Semi-dominant = only one copy matters
• Recessive = both must be mutant to see phenotype

• missense: change of one amino acid into another
• Nonsense: Change of one amino acid into the codon for stop
• Frameshift by addition: addition of one 'A, C, T or G'
• Frameshift by deletion: removal of one

Aristotle

The study of animal development

The fusion of the mature sex cells, sperm and egg which are collectively called the gametes

• Vegetal hemisphere: Bottom part where the yolk is. Provides food for the animal
• Animal hemisphere: Upper half of the egg

External

• External
• Haploid nucleus of egg (Female pronucleus) to merge with the haploid nucleus of the sperm (male pronucleus) to form diploid zygote nuceus.

A fluid filled cavity - the blastocoel

• Begins at a point opposite the point of sperm entry with a formation of a dimple called the blastopore
• Blastopore expands to a ring and cells migrating through blastopore become the mesoderm.
• Cells outside become ectoderm
• Cells in vegetal pole become endoderm

A rod of mesodermal cells in the most dorsal portion of the embryo

• Meiosis 1: Interphase, Prophase 1, Metaphase 1, Anaphase 1, Telophase
• Meiosis 2: Metaphase 2, Anaphase 2, Telophase 1

Beginning of neurulation

• 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

Fluorescein-Conjugated dextran

Embryos made from tissues of more than one genetic source

• Green fluorescent protein
• Altered gene called a transgene
• Give cells a virus so that they fluoresce

• Organs whose underlying similarity arises from their being derived from a common ancestral structure
• Example wing of bird and human arm

• 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

• Embryonic period (up to 8 weeks in humans)
• Fetal period (remaining time in utero)

Plastic bottles

• 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.

Histones

• 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.

Head and thorax formation

• 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.

• 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.

• 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.

The nucleus

While the nucleus controls cell fate, differential gene expression underlies cell fate specification .

• Transcription of genetic information
• RNA processing,
• Translation
• Protein folding

• 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.

• Usually bridges the enhancer and promoter regions.
• The mediator initiates a chromatin loop connecting the enhancer and promoter

• fibroblast growth factor
• Often work by activating a set of receptor tyrosine kinases called fibroblast growth factor receptors (FGFRs)

Cholesterol

Often created by the embryo to induce particular cell types and to create boundaries between tissues

3

• Proteins
• Patched

Smoothened

• Patched
• Smoothened
• Transcriptional activator

• 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

Frizzled

A transmembrane receptor that Wnt binds to

• B-Catenin
• Degraded

• 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

Dickkopf

• 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

The TGF-B family

• Nodal and activin
• BMP
• GDNF

They have 7 rather than 9 conserved cysteines

Bone morphogenetic proteins

• Bone formation
• Regulate cell division and apoptosis
• Regulate cell migration and differentiation

Specifying the different regions of the mesoderm and distinguishing the left and right sides of the vertebrate body axis

• Smad
• Ligand
• Receptor
• Receptors
• TGF receptor
• Smad
• Transcription factor complex
• Nucleus

It extends through the cell membrane and it's external factor contacts with Delta of another cell.

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

• Observation of cells
• Fate mapping
• Cutting up embryos
• Transplantation

• Gene cloning
• Gene expression analysis
• Gene loss of function
• Gene gain of function
• Protein localization
• Protein-protein interact
• Protein activity

• 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.

Knockout

Creating a knockout organism

• 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.

• Calcium Dependent adhesion molecules
• Critical for establishing and maintaining intercellular connections

catenins

• 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.

• A teratogen
• 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

By size

Strongylocentrotus pupartus

• Transparant
• Can watch cell division for the entire organism (very few cells)
• convenient and easy to raise in the lab

• Currently unable to make mutations of genes
• Much more limited scope of techniques can be done

• Hardy
• Inexpensive
• Transparent embryos

• Take a nucleous from an albino frog and inject that in an unfertilized embryo.
• New embryo is albino

• Bind to a ligand-binding domain that can activate an enzymatic activity
• Activates an active protein

Notch-Delta signalling

In Situ hybridization

• The proportion of the gene expression
• Does not give an exact numerical value

• Short generation time
• Four pairs of chromosomes
• High fecundity
• Easy anesthesia
• Salivary polytene chromosomes are highly duplicated and lined up
• first behavioural screens

• 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

• Activation of the zygotic genome in one of the following transition:
• MBT (Mid blastula transition)
• MZT (Maternal zygote transition)

• 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

• Regional identifiers
• Large portions of embryo are deleted
• Gene hypothesized to function in defining segment identity.

• Head region
• 3 Thoracic
• 8 Abdominal

13

• G1 Phase
• Transcription

Activation of Cyclin E

Segmentation

• Function to specify parasegmenta identity
• Affect embryo two-segment periodicity

• Regulate a subdomain of every segment
• Also function to define anterior vs posterior polarity within segments
• Specify cell identity within segments

Segment polarity genes!

• 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

Polarized microtubules

Anterior fate

• Normal development has bicoid at the anterior end of the embryo.
• An embryo will have two tail regions in a bicoid deficient mutant

• 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.

• Nanos is on the posterior side of the embryo
• Inhibits Bicoid

Not until after the 13th nuclear division

Gurken protein enters the nucleus and ventralizes the cell

• 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.

• 7/14
• 14/14

• No, because the step from RNA to protein is regulated
• Can create a gradient

RNA, which makes proteins

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.

Nanos in the posterior region of the embryo inhibits hunchback protein and allows for abdominal formation.

To examine regulation of pair rule genes

The Posterior region

• Anterior
• Posterior

Individual cells

• Wg gene
• 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

• 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

• Tolloid
• Decapentaplegic
• Dorsal
• Ventra

• 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

• 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

• Fly with extra set of wings
• Example of homeosis
• Loss of Ubx gene function

• 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

DNA binding domain

All on the same chromosome next to each other

• 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.

A homeotic selector Gene

• Sperm contacts jelly layer
• Acrosome reaction
• Digestion of jelly layer
• Binding to vitelline envellope
• Fusion of acrosomal process membrane and egg membrane

• Capacitation
• Occurs in female reproductive tract

Chemotaxis

• This is needed because of the external fertilization process
• Sperm-activated peptides are released along with the eggs.

• A protein called bindin.
• Species specific

• Bindin binds to the surface of the egg
• Actin microfilaments extends by actin polymerization.

Bindin

• Two blocks
• 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.

• Mesolesical egg - more yolk at ventral pole
• Displaced radial cleavage
• Cells at the bottom larger

Vegetal pole is at the bottom => all of the yolk to support the embryo at the animal pole (top)

• 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

• Three tissue layers
• Endoderm
• Ectoderm
• Mesoderm

• 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.

Primitive gut

• 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).

• 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.

• Cells involute and ingress (fold inwards)
• Move toward the midline of the embryo/egg

• Cells of the epiblast and hypoblast intercalate to form a thickening.
• The cells converge and extend anteriorly. (move up)
• Dorsal side

• Animal cap cells => ectoderm
• Marginal cells => Mesoderm
• Vegetal cells => endoderm

• Ectoderm
• Mesoderm
• This is done by factors released from vegetal cells - usually when the marginal cells have been removed experimentally

Growth factors

Growth factors

The dorsalmost vegetal cells in frogs that are capable of inducing the organizer.

• 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

• 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.

• Cells that are grown in a dish
• They grow without adding a growth factor
• This suggests that they secrete their own mesoderm inducing signal

XTC cells create growth factor - this was important in diagnosing what the specific mesoderm inducing signal is.

• through cell culture work. Both XTC and Actin A have mesoderm inducing factor
• However Actin is not the mesoderm inducing factor.

• 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.

• A transcription factor that is important in activating the mesoderm inducing factor
• Regulated nodal related
• Nodal related is the mesoderm inducing factor

• Nodal related
• Xnr in frogs (Xenapus nodal related)

• Large embryo
• Allowed us to find the mesoderm inducing factor (nodal related)
• Also large number of embryos - a very large amount of source material.

• Derriere and Nodal related genes
• Induces them so that they can form mesoderm

Yolk syncytial layer

Holoprosencephaly

Cyclopia in sheep

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.

• Tetraploid
• Takes a long time to reach maturity

Very powerful methods in an unbiased method to ask the embryo what genes are important for specific functions/characteristics

• Cyclops
• One eye pin head

• They lack mesoderm
• Gene of importance is squint

• Found in the group of nuclei between the yolk syncytial layer and the embryo
• In zebra fish
• Remove mesodermal induction
• Increased mesoderm induction

• 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.

• 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.

Starts as early as the 2-4 cell stage.

Cortical rotation

• Through transplantation experiments
• Identifying the Nieuwkoop center and dorsal organizer
• Look for localized proteins and mRNA that move during cortical rotation

UV

The dorsal side of the embryo

• 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

transcription

• 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

• Chordin, Noggin, Follistatin and Xnr 3
• Specifies the dorsal region of the embryo

The zebrafish shield

The loss of dorsal structures

The 4-cell stage

• It is a BMP mutant phenotype
• over expression of swirl causes ventralization

• 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.

• Forebrain: Telencephalon and Diencephalon
• Midbrain
• Hindbrain: Metencephalon and Myelencephalon

• Wnt binds to it instead of the receptor (Frizzle)
• When there is a lot of Frzb, there is no Wnt binding

The cells that make the anterior neural border

No development of anterior markers in neural tube development

• No development of posterior markers
• Lots of anterior neural tube development

• Frzb is expressed in the anterior region of the neural tube
• Wnt is expressed in the posterior region of the neural tube

Segmental

• A hindbrain segment of an embryo
• Hox genes show exquisite segmental expression pattern in the hindbrain

• A molecule that contains a multiple nucleotide analogue
• Derivative of oligonucleotide

• 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

Vitamin A

• To label a portion of the embryo
• To study gene regulation
• To overexpress/ectopically express gene (ectopically = different region)

• 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

It is flipped in comparison with other animals

• 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)

• important in the retinoic acid pathway
• cyp26 is located in the anterior region.
• aldh1a2 is located in the posterior region