Chp 1&2: Development Flashcards
Cleavage
- process of creating blastomere (smaller and smaller cells due to division) ->blastula
- leads to a cavity in all organisms called the blastocoel/blastopore
Fertilization
Brings together 2 genomes
Ex: frogs have chemical signal that tells them to produce gametes
Females->produce yolk->zygote
Gastrulation
-gastrula
-germ layer formation (3 layers):
Endoderm: lining of intestines/lungs
Ectoderm: skin, nervous system
Mesoderm: skeletal, muscles, parts of organs, blood, becomes gametes
-moving of the blastomeres resulting in the germ layers
Organogenesis
-interactions, rearrangements, migrations
Ex: notochord= works as a signal for different tissues -> nervula
Metamorphosis
- larval stage (non sexual) to sexually competent adult
- different depending on group of organism
Germ cell - Gametogenesis
-tends to be isolated during development due to the different signals
Development
Aristotle 350 BC- used chickens
Limited to multicellular except in yeast cells
Embryology= old name
Continues past gestation (humans late 20s)
3 approaches to dev bio
Anatomical
Experimental
Genetic
Anatomical
Blastomeres-cells & fate
Comparative embryology - dev diff
Evolution
Teratology- teratogens: result in birth defects
-observe deformities to inform what went wrong
Mathematical- pos. & neg. feedback
-chemical
Ovoviviparity
Eggs w/ yolk
Hatch internally
Oviparity
Egg layers: birds, frogs, insects, monotremes
Viviparity
Placental mammals
After Aristotle …
Nothing happens bc of religion
- William Harvey (1651)
- late 1600s: Enlightenment->science kicks in
William Harvey
“All animals are from eggs”
NO spontaneous generation
Tried to find the mammalian egg
-used deer
1672: Marcello Malpighi
Microscopes
-microscopic accounts of chick
How dev. occurs?
-epigenesis -> organs from scratch vs. preformation -> everything is already there, it’s just miniature
-no cell theory, so no limits on how small something was
Kasper Wolff
Supports epigenesis
Watches late tissue formation
1820s: several German scientists
Germ layers
Microscopes get perfected
-new staining techniques that allowed them to see small structures
*ectoderm, endoderm, mesoderm
-interaction was critical
~of the layers: to know what they’re purpose is
-relationship among early embryos and their structures across species
-the closer related… The longer it takes to distinguish embryos (humans + chimps)
-as dev progresses, characters go from generic -> specific
2 kinds of cells
Epithelial - sheets
Mesenchymal - wanderers
Morphogenesis
Due to a limited amount of cell activities of cell activities
*where they go + how much they divide can have cell shape change
Mesenchymal-> epithelial-> tube-> sheet
Fate mapping
-almost the point where medicine comes in
-end up with cell lineages
Each cell is a daughter
-tunicates
Look like tadpoles as larvae -> bag of goo
Cytoplasm had diff colors BC of germ layers
Test fates by removal of mesodermal cells
Genetic Labeling
1920: Hilde Mangold + Hans
Chimeras
Chimeras
2 genetically diff species mixed
-1st ones done on newts
-chick + quail: easily identified cells
>Condensed dna
>have specific antigens
>neural crest cellsTransgenic chimera
Today: put in green fluorescent + protein (GFP)
Life Cycles (review)
-Fertilization>hatching=embryogenesis 1 cleavage 2 gastrulation 3 organogenesis 4 gametogenesis 5 metamorphosis
Cleavage
Rapid mitoic division
Zygote> blastula
*volume stays the same
Gastrulation
Cell movement resulting in germ layers
Organogenesis
- formation of organs
- a lot of cell communication
- some organs = multiple germ layers
- cells migrate
Metamorphosis
-maturity> related to gametogenesis (“not ready”) where germ cells are set aside for reproduction + protected
*fated of cells depends on what they’re next to
>all other cells are somatic (46 chromosomes, go towards creating the body)
Frog Life Cycle
-when conditions are good (enough sun, good temps) females will make yolk in the liver which is packed into the eggs
-Fertilization
>germ cells move into the gonads from a hiding spot in the endoderm
-eggs come from oognia
-sperm comes from spermatogonia
Mitosis (in frog)
Germ cells from oognia or spermatogonia
Primary oocyte spermatocyte> meiosis(2n->1n) > homologous chromosome pairing shuffling daughters = haploid> 1n - secondary oocytes
-in the egg meiosis stops early + sits in stage of haploid
Until it reaches sperm…egg complete
-nucleus in the egg = “pronucleus”
-nucleus + pronucleus combine to form zygote (2n)
-as sperm hits egg> cytoplasmic rearrangement (change of color, movement)
Cleavage (in frog)
- volume remains the same
- blastula= 10s of thousands of cells
- animal pole divides quickly (sm. cells)
- vegetal pole is slow (large cells)
- blastocoele forms (fluid cavity)> animal pole
Gastrulation (in frogs)
- sperm entry defines dorsal surface of organism
- as cells migrate in through blastopore (lip, gray crescent) they become mesoderm
- the cells outside > ectoderm
- blastopore becomes neural groove eventually
- the large yolk-filled cells in the vegetal pole> become endoderm
Organogenesis (in frogs)
Mesoderm in the most dorsal
-neural groove
-neural tube> nervous system
Ectoderm above notochord becomes tissue in your spine
Embryo> neurula
Ectoderm grows over the neural tube (birth defect if doesn’t grow)
Mouth & anus form
Muscles form (dev point when start moving, where it hatches)
Cell specification
“Presumptive tissue”
1) specification: if put in a neural environment, no signals> follows fate
2) determination: follow fate regardless of environment, irreversible
Ex: tunicate- in the cleavage stage, the 1st blastomeres determine fate
Types of specification
1) autonomous >specific blastomeres translates to specific tissues/parts determined by cytoplasmic constituent
*proteins- transcription factors
*mRNA
>fates are invariant
>most invertebrates
2) conditional specificity
-all vertebrates a few invertebrates
-fate is determined by your “friends”(environment)> happens a little later
-little invariant fate assignment
-can frequently switch fates (good thing)
-cell rearrangement> migrations> specificity: *development gets regulated which allows cells to acquire a variety of characters
3) syncytial specificity
-mostly insects (all insects do this)
-localization of cytoplasmic constituants
Genetics
-visible mutations on the chromosome lead to the nucleus being determined as holding the info/DNA (variable)
Cell Fates & Cloning
-do cells become differentiated?
-easy in the 1950s (thought of in 1890s)
>remove nucleus from oocyte, remove the donor nucleus from cell, transplant donor into oocyte
Somatic nuclear transfer (cloning)
1956: King + Briggs
-used tail bud nuclei for a SNT
>result: nothing
-used germ cells nuclei
>result: frog
*establishes that the DNA is being modified/responsible
DNAs fate= determined
Clones= twins
1975: serial Transplantation
Took adult frog foot web cells
>ectoderm (skin, nervous tissue)
>very specialized
-do the SNT>embryos>gastrulation
-go into blastula (pregastrulation)>take nucleus + transplant into another oocyte which survives up until tadpole stage then dies
*proved reversible differentiation is potential in all cells
1997: Dolly (sheep)
-G1 stage: mammary cells (diff breed) from one sheep
-enucleated an oocyte (in 2nd meiotic phase)
-fused the cells w/ electroshock (used to get the egg cell to believe it was fertilized) in this case>membrane fusion
-did process with 343 cells
>result: 1 Dolly
Totipotent
Creat every cell possible
- no genes have been lost/mutated during differentiation
- phenotype is not identical
How do you turn on/off genes?
1) every cell w/ a nucleus (RBC don’t have nucleus) is the same DNA
2) unused genes are not destroyed so potential for expression exists even if everything’s shut off
3) only a small % of the genome is expressed in a cell
>RNA should be specific
Fruit fly gene expression
-used polytome chromosomes
-DNA replication but no mitosis 2^9 vs. 512
-150 times the normal amount
-found that regions of the chromosome
>”puff out” when transcribed
-DNA rep. found in salivary glands
Other gene expression studies
- confirmed point 3> DNA RNA hybrids
- label DNA or RNA seq of interest and put them in w/ RNA isolated from cell
Diff. Gene Expression Techniques
RT PCR
RT= reverse transcriptase (virus) PCR= polymerase chain rxn
- take RNA + turn that into DNA using RNA virus (RT)
- in the PCR, polymerase is used
Diff gene expression technique
cDNA
Complementary DNA sequence bound to a plate
Insitu hybridization
- labeled anti sense RNA (radioactive or dyed)
- look for areas of expression
- can look at quantity of expression
Transgenics
-allows you to look at gene function during normal dev
-doesn’t damage dev of organisms
-can inset cloned DNA into cells
>microinjection
>transfection -low intake
>electroporation -speed intake by shocking EX: sodium shock
Transposable Elements
"I am legend" movie
-retroviral vectors (Gene therapy)
>near 100% insertion (you'll get results)
>replace viral proteins w/ phyload
-marker product (GFP)Chimeras (mice)
- take a blastomere from mouse A + put it in mouse B early, it contributes to all tissues
- took from inner cell mass then they were totpotent
Gene Targeting
- from knockouts
- if they have 1 good copy then the organism will develop
- if knockouts are crossed then you’ll get heterozygotes
- break gene l, microinject, then cells swap out bad gene for good gene
- Bone morpho protein/BMP 7 Knockout: the heterozygote develops normally + the knockout has no eyes or kidneys
Newer: Antisense RNA
-inject a large amount of it into cell which makes the cell destroy both sense and antisense forms of RNA
>cell does all the work
-Morpholino Antisense (permanent poison)
>hard for the cell to destroy RNAs
>double stranded RNA triggers an antiviral response
Differential Gene Expression
~25,000 protein encoding genes
-dev genetics
>how do you take same genes + create diff cells/tissues
>how do you go from genotype -> phenotype
*diff from cell or molecular bio is the level of interest
>want to find out how the cellular processes function on a tissue/cell level
Gene Expression
-proteins lead to everything else except ribosomes
-occurs at multiple levels:
1) Diff Expression -> RNA
2) Selective nRNA processing
-which nRNA->mRNA goes to cytoplasm
-intron + exon splicing (antibodies)
3) Selective Translation
>RNA to protein
4) Differential Protein Modification
>degradation
diff signals to express here AND for how long
>activation
Chromatin (active or repressed)
> complex of DNA + proteins (histones)
-nucleosome= basic unit of chromatin-> DNA (2loops) wrapped in 8 histones
~140 base pairs
Chromatin
String of nucleosomes which are connected by H1 linker proteins (histones)
Histone
Cause nucleosomes to get wound tight
-organize DNA strands into nucleosomes by forming molecular complexes around which the DNA winds.
H1 confirmation represses transcription
(take yarn off ball when knitting)
-when H1 linkers are removed, it allows transcription factors to get in there
Nucleosomes and H1
Limit access to DNA
-default condition = repressed
-specification/diff. -> cell/tissue specific change to the repressed state
> use histone acetyltransferase(can add acetyl groups)
Methyl + acetyl groups
Location: on tails
- tighten or loosen nucleosome
- remove methyl= loosen
- remove acetyl= tighten
- histone methyltransferase adds methyl groups (tightens)
Introns + Exons
Introns= loose Exons= exits nucleus/keep
What is “the gene”?
*promoter: polymerase binding/initiation
>upstream (5’)
*transcription + translation termination helps form poly-A tail
-before leaving nucleus:
>nRNA will get a 5’ cap of methylated guanine
>reverse polarity so there’s no 5’ end
*poly-A tail
>needed to bond to ribosome
*3’ poly-A tail
>protects from exonucleus
Promoters & Enhancers
-determine when + where genes are expressed
Promoter
Upstream- bind polymerases and also transcription factors
Basal transcription factors (TFs)
Necessary for polymerase binding
Ex TATA binding protein (TBP)
TFIID
Fraction of a cellulose extraction
- foundation of the complex of proteins needed for initiation
- stops nucleosome formation
TFIIA
(Larger protein)
- binds w/ TFIIB so that RNA polymerase can bind
- factors E, F, H release RNA polymerase + unwind helix (like helicase)
- regulated by TBP Associated factors
TBP Associated Factors (TAF)
Can help modulate activity of the RNA polymerase activity-bound upstream of promoter + is tissue specific
Enhancer
Control efficiency and rate for a specific promoter
*importnat in tissue specific exp.
-can be 1000s of base pairs away but on the same chromosome (limit)
-5’ (upstream) or 3’ (downstream)
-function= combine w/ transcription factors
-nothing can bind to it
-removing methyl groups
-most genes require enhancer activity
-multiple enhancers + multiple TF can all work on 1 gene
>want to have enhancer to work the right way + same place
-enhancers can also inhibit
-mixed activity
Transcription Factors (TF)
-proteins that bind to enhancers (DNA seq) and promoters
-activate or repress (variably) a lot or little
-grouped into families
>small amino acid changes, change binding
Hox Genes
-codes for TF
-axis formation
Pou= pituitary and neural fates
Lim= head
Pax= neural and eye dev
-basic structure= combination of Leucine zippers, Helia loophelia, zinc finger
3 TF domains
1) DNA binding domain
2) Transactivating domain
>important in activating/suppressing transcription
>binds to proteins (TFIIS modifying histones)
3) protein-protein interaction domain
>other TFs or TAFs(transcription associated proteins) can come in and modify activity
Ex 1 of TF domains
MITF
-TF that’s active in ears and pigment cells
-heterozygote (1 functioning form of MITF)
>deaf, multi colored eyes, white forelock
>helix loop helix structure
*protein-protein domain allows it to dimerize w/ MITF
*can then bind to DNA
*transactivating domain binds w/ a TAF (acetyl transferase)
Ex 2 of TF domains
Pax 6
- active in mammalian eye, nervous system + pancreas
- contains 2 DNA binding domains
- pax 6 binding seq. found in lens gene enhancers and endocrine cells in pancreas that are important in insulin/glucagon (Diabetes)
- can activate or suppress
- pax 6 + sox 2 have to be present together for lens dev (eyes won’t form)
- sox 2 is only present in ectoderm that’s been exposed to optic vesicle
3rd Binding site of Pax 6 TF domain
- repression or activator
- critical in stopping lens formation outside eye
- when expressed it does a positive feedback (becomes permanent enhancer)
Transcription Factor Cascades
-TFs are activated by TFs
>Pax 6 is turned on by TF: MBX which is expressed in late gastrulation
-Activation of TFs
>may need a signal to be competent
Silencers
Repressed that repress transcription
Ex: neural restrictive silencer enhancer (NRSE)
>causes promoter only to be active in neurons
-expressed in all other cells
-works as deacetylase
Methylation
-DNA methylation
>how expression becomes stable -> adulthood
-promoters can become methylated
CpG seq because cytosine gets methylated but only if followed by guanine
-methylated cytosines stabilizes nucleosomes
>results in TF not binding
Genes during development
-genes only active in sorry or some active in egg
-methylation of CpG
-imprinting
>disadvantage: lethal alleles exposed
How does methylation stop transcription?
-interaction w/ histones
-methylated DNA attracts enzymes that further methylated DNA
-methylated DNA stabilizes nucleosomes beyond stabilized methylation of histones
*Mecp2 (protein)> selectively binds methylated DNA and also binds deacytelases
(H1 linker histone) ->attracted to methyl DNA
Clones
Compare to normal formation -> methylation is messed up
Normal DNA methylation
maintained during mitosis by DNA methyl transferase (1 strand methylated + the copied strand will be methylated)
Loss of methylated transferase (mice)
Small shortly after birth, die because of multiple malignant tumors
Insulators
Seq that binds proteins “insulators” stops activation of adjacent promoters often located between enhancers and promoters
Dosage compensation
Inactivation
Ex: X chromosome
X converted to heterochromatin
Heterochromatin remains condensed
Replicates later than the other chromosome
Forms Barr body
*if not shut down; death because only want same X chromosome shut down in every cell
*early inactivation critic
Shut down isn't always 100%
-15% could still be active (alleles)
-germ s will cause reactivationInactivation
= a chain reaction:
MeCP2 > methylated DNA
Stabilized nucleosomes > deacytelases
EZH2 > methylates DNA & histones
H1 highorder folding (nucleosome)
*all of this initiated by RNA seq that never leaves nucleus
-methylated promoters
Differential RNA processing
(DNA>nRNA>mRNA>proteins)
After transcription> translation
1) mRNA (introns, cap, tail)
2) move from nucleus> cytoplasm
3) translation> post-translation changes
-diff in cells=diff in proteins
-same pool of RNAs but diff sets make it to the ribosomes (called censorship)
-splicing: exons + introns
*combine multiple exons differently
*nRNA contains introns
>pre mRNA
*by processing diff subsetsExample of differential RNA processing
Sea urchin blastula vs gastrula
- more genes are transcribed than expressed
- the stuff not sent out = degraded within nucleus
Alternate RNA splicing
-normal vertebrate RNA has many introns + exons that get spliced together> shorter seq
-splice sites (introns) decide if it’s cut out or left
-spliceosomes= bind to splice sites
>made of combination of RNA + protein (splicing factors)
-diff spliceosomes = diff results
-splice site recognition is 5’
Alt. RNA splicing example
Tropomyasin
-1 gene for Tropomyasin
BUT diff kind (slight modifications) of Tropomyasin in brain, liver, skeletal muscle, etc.
Multiple protein forms
*called: splicing isoforms
Alt. RNA splicing example
BC1-X
1 form inhibits apoptosis
Other fork induces apoptosis
Alt. RNA splicing example
Fruit flies
1 gene in drosophila has 38,016 potential isoforms
*genome and protenome are not equal
Differential Splicing
Enhancers etc.->DNA->RNA
-diff proteins recruiting to splice sites
>changing splice activity
Translation
Controlling protein creation
- mRNA longevity> stabilized RNA leads to more protein
- stability is a function of poly A’ tail
Untranslated region
UTR
(Not turned into proteins)
-determines length
-when experiment alters UTR it increases or decreases the half life of mRNA
-caesin mRNA: half life= 1.1 hours (during lactation= 28 hours)
>protein found in milk
Oocyte translation
Make and store mRNA that is used much later after fertilization
- necessary for chromatin, membranes, cytoskeleton components
- early cell divisions rely on this
Bicoid & nano translation
(Insects)
Localize (mRNA) to specific parts of cell
Translation regulation
-tends to be negative
-default state= on
-5’ cap 3’ tail is where this occurs
>important in ribosome binding
*no cap/tail = no translation
mRNA
Circular
5’ held to 3’ by proteins *important in unwinding double stranded RNA
3D structure
Ex of mRNA
EIF4G
Binds 3’
Binds ribosome
Ribosome binds 5’ initiation factor
Ex of mRNA
EIF4G
Binds 5’ cap
Interacts with G
So oocytes produce RNAs without cap
-fertilization >capping
Cytoplasmic localization
3’ UTRs
-vegetal localization (yolk rich)
-bicoid (anterior) nanos (posterior)
>if 3’ UTR from bicoid and add to RNA it knows where to go
Post translational
Structural
Enzymes
Phosphokinase
TF
For many proteins to become active…
Need something else: -dimer >MITF - hemoglobin -cleaved - insulin -ribosomes - tubules -ion binding
