Developmental Biology Exam 4 Flashcards
Dorsal
Top
Ventral
Bottom
Posterior
Side towards pinky
Anterior
Side toward thumb
Proximal
Close to body
Ex. Upper arm
Distal
Far away from body
Ex. Fingers
The limb bud
Needed to form a limb
Forlimb field
Emerges from lateral plate mesoderm
Middle to bottom of organism laterally
How the limb bud emerges
Limb bud comes from lateral plate mesoderm
Mesenchymal cells are filling the limb bud
Proliferation of mesenchymal cells in lateral plate mesoderm causes limb bud to bulge outward
Going to be limb field
Two things needed for limb development
- Limb bud induction and outgrowth
- Cell fate specification and axis patterning
How is early limb bud induced?
Morphogen gradient between Fgf8 and Retinoic acid (RA)
Fgf8 is expressed at the head and the tail end of the organism.
RA is expressed in the center of the organism around the somite area
Fgf8 and RA inhibit each other.
Wings:
RA activates the txn. factor Tbx5 which causes the forelimb field
Tbx5 induces Fgf10 which promotes proliferation of the lateral plate mesoderm which is on both sides of the organism
Wing formation x2
Legs:
It is not known what causes the expression of Tbx4
Expression of Tbx4 to cause Fgf10 expression to get proliferation of the lateral plate mesoderm on both sides of the organism
Leg formation x2
Where does proliferation of the limb bud happen?
In the progress zone
Have proliferating mesenchyme
Apical ectodermal ridge (AER)
Surface ectoderm
Thickens on the distal side of the limb bud
Epithelial thickening at the distal edge of the limb bud
Model for forelimb development
RA causes expression of Tbx5
Tbx5 causes expression of Fgf10 which causes proliferation and expression of Wnt3a
Wnt3a cases expression of Fgf8 which leads to Fgf10 expression
Fgf10 has a positive feedback loop with Tbx5.
Fgf8 has positive feedback loop with Fgf10
Wnt3a and Fgf8 are expressed between the AER and the progress zone (PZ)
This suggests the apical ectodermal ridge (AER) is responsible for allowing the proliferation to happen
What is the signaling center for limb buds?
The AER
It is necessary to keep limb bud growing as it extends
Secondary signaling allows growth to continue as it gets farther away
Evidence:
When the AER is removed, limb development ceases
When AER is replaced with an Fgf bead, a normal wing forms
How is axis formation established during limb development?
Morphogenetic gradients:
RA and Fgf8
Proximal to distal
Shh on posterior side
Anterior to posterior
Wnt and BMP
Dorsal to ventral
Proximal-distal limb patterning
Proximal end expresses R
Threshold of RA is required for “stylopod” development
Distal end expresses Fgf8
Threshold of Fgf8 for “autopod” development
The evidence of proximal-distal limb patterning by RA
- Collect undifferentiated PZ cells from different stages of limb bud growth
- Transplant these cells into a young limb bud host
- Skeletal structures show being farther away from RA depend on the position/age of our donor cells
Closer to proximal end creates zeugopod and farther away creates autopod
Can repeat experiment but treat transplant tissues/zone with RA
Skeletal structures follow pattern. Seems to have shifted everything more proximal
Order of proximal distal limb patterning from proximal to distal
Stylopod, zeugopod, autopod
How is the lack of RA causing different tissue types in proximal-distal limb patterning?
How genes!
What genes specify identities along proximal-distal axis?
How genes
Hox loss of function leads to loss of zeugopod bones
Early limb control region (elcr)
High RA activates that region
Apical ectoderm ridge (AER)
Thickening at distal edge of limb bud
Part of limb bud
Progress zone (PZ)
Proliferating mesenchyme
Part of the limb bud
Zone of polarity activity (ZPA)
Mesodermal cells in limb bud posterior that specify anterior/posterior axis and all fates
Part of limb bud
Shh is expressed in posterior of limb buds and limbs
What happens if you transplant ZPA to the anterior side so you have posterior and anterior ZPA?
Graft a second ZPA onto anterior of limb bud
Get duplication of the normal digits
The duplication is a mirror image digit pattern
What happens when you remove Shh from posterior limb bud?
Only most anterior digit forms (d1)
Suggesting it might not need Shh to form
Maybe it gets less morphogen of Shh since it is further away
How to remove Shh from limb bud?
Make a oligozeugodactyly (ozd) mutant which lacks Shh function in the limb
Deletion in ZRS
Results in loss of Shh in the limb bud
What determines digit identity?
Gradient of Shh
Shh expressing cells are in the ZPA
Creates a gradient to the anterior side
Concentration and length of exposure is out digit identity
Shh has auto one and paracrine factor in a posterior-anterior gradient
What activates Shh?
Called Temporal Colinearity-“Phase I”
Posterior Hox expression
It activates Shh via ZRS
This is how we get Shh is in ZPA
ZRS is enhancer that Hox genes activate. The enhancer then activates the Shh gene
Temporal Colinearity-“Phase II”
Posterior Hox genes express Shh, gradient acts on GCR (global control region) of gene to activate the GCR
GCR expression in reverse direction. Flips Hox so areas getting most Shh
Creates a nested pattern which leads to digit identity
How does the ZPA support limb growth?
Shh is expressed which causes the expression of Gremlin.
Gremlin presence keeps Fgf8 around to get continued limb growth. Therefore it inhibits BMP since BMP inhibits Fgf8
Fgf8 supports Shh presence which therefore is a positive feedback loop to get more limb growth
This maintains A/P patterning
There is a negative feedback loop when Fgf8 inhibits Gremlin
How limb growth ends
BMP signals the end of limb growth
1. Fgf8 levels accumulate and at high levels, Fgf8 inhibits Gremlin
2. ZPA stays in one spot as limb bud grows away. Therefore Shh can only go so far since it is made by cells in ZPA region
Shh and Gremlin are further away from AER (Fgf8)
Less Gremlin nearby, BMPs can begin to inhibit Fgf8 in the AER
Therefore limb growth stops
Dorsal-ventral axis
Wnt is on dorsal side, BMP is on ventral side
These morphogens specify dorsal-ventral patterning
Dorsal ectoderm setting up D/V polarity
Opposing Wnt 7a and BMP signals determine D/V fates
Wnt 7a activates txn. factor Lmx1b in underlying mesenchyme
Lmx1b is a txn factor for dorsal fates
What happens if there is no Lmx1b for D/V fate?
The limb is ventralized
Roles of BMP in limb development
- Stop limb growth
- Indirectly inhibits ZPA/Shh
- Promotes ventral fates, can inhibit dorsal fates
How are limbs sculpted?
Cell death
BMP induces apoptosis
Therefore BMP is important for sculpting digits/limbs
Gremlin inhibits BMP to stop apoptosis
What is one of the leading causes of death for infants and children in the United States?
Congenital Anomalies
Paths to abnormal development
- Random events/bad luck
- Genetic mechanisms
- Environmental factors - teratogens
Pleiotropy
Several affects from one gene
Mosaic pleiotrophy
Single gene causing different phenotypes by independent mechanisms
Effects of mosaic pleiotrophy
KIT is receptor that gets P’ed
When not expressed in embryonic germ cells:
Loss of function leads to sterility
When not expressed in HSCs:
Loss of function leads to anemia
When not expressed in Melanoblasts:
Loss of function leads to albinialism
Genetic heterogeneity
Mutations in different genes may cause the same phenotype
Ex. Formation of one central eye instead of two (Cyclopia)
Get LOF mutation in Shh (or its regulatory region)
Defects in Shh pathway or its downstream proteins
Defects in ability of Shh to be a defusable morphogen
Normal eye specification
Eye field (EF) forms in the anterior neural plate
EF expresses Pax6 txn factor
Shh in prechordal plate (aka. Notochord) inhibits Pax6. Therefore you get two EF’s which forms two eyes
Teratogens
Environmental factors/external agents that cause birth defects
When is the fetal susceptibility to teratogens the highest?
During the embryonic period
Effects of alcohol on the fetal brain
FAS brain is smaller, underdeveloped, less neural projections in posterior, small, smooth brain, low surface areas, slower mental processing, on the spectrum
Alcohol effects on baby mice
Small nose, abnormal upper lip, smaller brain thought to be caused by cell survival
Less adhesion of cells with more alcohol exposure
Defects in cell adhesion, cell migration, cell division (neural crest progenitors must migrate and proliferate) and cell survival
What alcohol does the mouse embryos
Death of cells since alcohol induces ROS
Alcohol + NAD+ —> Acetylade + NADH
NADH is an electron carrier which makes it more of a free radical
Teratogenic effects of retinoic acid
Vitamin A (retinol) —> retinaldehyde —> RA
Beta- cantere causes the same reaction
RA and vitamin A are teratogenic during weeks 3-5
[RA] gradients are carefully controlled/regulated during development
Excess RA triggers negative feedback loop that equals a long term decrease in RA levels
Pathogens to abnormal development
Random events: bad luck
Genetic Mechanisms: mutations, aneuploidy
Environment Mechanisms: teratogens
Possible mechanisms for endocrine disruptors
- Mimic natural hormones
- Inhibit function of hormones
- Interfere with synthesis transport or elimination of hormones
- Sensitive to hormone signaling
DES chemical
Mimics natural hormones
Designed to do same thing as estrogen
Is an estrogen analog
Binds and activates estrogen receptor
Vinclozolin
Fungicide
Used to treat fungus
Binds and blocks testosterone receptor
Inhibits function of hormones
Atrazine (herbacide)
Promotes estrogen synthesis at expense of testosterone
Interferes with synthesis transport or elimination of hormones
EPA
Mice injected with EPA in utero enter puberty early
called “precocious puberty” in mice and humans
Sensitive to hormone signaling
BPA
Bisphenol A (BPA)
Steroid hormone analog (estrogen)
BPA strongly binds estrogen related receptor gamma (ERR-gamma)
Will cause activation of downstream targets
Leads to aneuploidy
Random chromosome alignment on meiotic spindle
Found to cause birth defects and miscarriages in humans and monkeys
Transgenerational inheritance with vinclozolin and rats
The rats injected with this chemical had the BPA in this chemical alter DNA methylation of over 100 genes
Passed on through sperm DNA
F1 males exhibit testicular defects with low sperm production and motility
F2 males exhibit same phenotype
F3 males exhibit same phenotype
F4 males exhibit same phenotype
Antisocial behavior in mice with anti-butt sniffing
Aso a decrease in oxytocin in injected mice which makes them sad mice. Not happy :(
BPA and cancer susceptibility
More buds/branches/growth in mammary glands
Overgrowth in mammary glands
These are precancerous indicators
Cancer origin theories
Somatic mutation theory
Tissue organization field theory
Somatic mutation theory
Normal one
Cells are quiescent (not proliferating) mutations allow autonomy (over-proliferation, migration, etc…)
Tissue organization field theory (TOFT)
Suggests that cells have ability to do what they want all the time (are proliferative or migratory), but communication with/within other tissues prevents that bad behavior
Therefore cancer would happen where this communication is severed
Evidence for TOFT
Aggressive metastatic melanoma cells were injected into a 2-day chick neural tube
Cancer cells can integrate into neural crest
Some are populating head to be facial/cartilage cells. Are being normal cells
Some are being normal and integrating into environment
Differentiate normally
Were kept in check by tissue environment
Testing TOFT in rats
When carcinogen is injected into only epithelium, there are no rats with tumors
When carcinogen is injected into only mesenchyme, there are rats with tumors
When carcinogen is injected into mesenchyme and epithelium there are rats with tumors
This supports the theory since epithelial cells do not get cancer since they are monitored by their neighboring cells when mesenchyme cells are not as close to their neighbors and therefore not as well regulated.
Descent with modification
Darwin with natural selection
1. All living to bugs are related
2. Organisms have changed over time
Explained these two points via natural selection
Example of descent with modification
Horse feet
Feet changed from multiple digits to one hoof
They grew taller
We know this because of fossil record
To see how single hoof was developing via examining hoof fossils: found all digits are still there. Other ones fuse into one big hoof
Evolution through developmental change
- Concept of modularity
- Concept of molecular parsimony
Concept of modularity
Idea organisms developed from a system of modules
Concept of molecular parsimony
Organisms use the same genes (tools/tool kit)
Modularity
Development as a system of modules
Modules
Segments
Cell lineages
Morphogenetic fields (limb bud, organ field)
Signal transduction pathways
Enhancers (plus other regulatory regions)
Enhancer modularity
There can be multiple enhancers for a gene since genes can be expressed in different places (heterotopy) through activating/adding enhancer region in gene
Example of enhancer modularity
Pilose pads on lizard are sticky hairs that allows them to climb up walls. Is on their hands and their tail
Same gene as that one is expressed on walder bird and causes them to have stripes
Enhancer modularity: threespine stickleback
In marine fish there are enhancer regions which cause the transcription of Pitx1 gene which makes spines. Have thymus enhancer, hindlimb enhancer (pelvis) and the sensory neuron enhancer
Freshwater fish are different and only have the thymus and sensory neuron enhancer. Do not have the hindlimb enhancer. Therefore the freshwater species have lost pelvic spines since the hindlimb enhancer is wanted
Module recruitment: elytra
Module for exoskeleton development that has been moved/recruited to make new structures
Elytra is a hard outer “wing” of beetles
Ex. Beetle has both hind wing and forewing (elytra)
In dragonflies the elytra is wanted and the fly elects to duplicate the hind wing to have a forewing and hindwing that are the same
Fly has FW but HW is haltere. The haltere is a stabilizing structure for the fly
Molecular parsimony
Organisms uses the same “small toolkit” during development
Examples of molecular parsimony
Paracrine factors (Wnt’s, Fgf’s, RA’s, and BMP’s)
Adhesion molecules
Txn. Factors (Pax6 for eye development, Hox genes for A/P patterning)
Homology
Homologues- derived from a common ancestor
Orthologues
Homologous genes between species
Paralogues
Homologous genes within one species (via gene duplication)
Mechanisms of evolutionary change
Heterotopy, heterochromy, heterometry, heterotipy
Heterotopy
Changing the place of gene expression
Heterochrony
Change in timing of gene expression. Maybe same place but different amount of time/different period
Heterometry
Amount of gene expression of a gene
Heterotipy
Change in type/function of gene, change in genes function being expressed
Interdigital webbing is an example of
Heterotopy because there is a difference in location of BMP inhibitor between a mouse paw and a bat wing
Digit length is an example of (mouse paw vs. bat wing)
Heterochromy- difference of timing
Heterotopy- differences in location
Heterometry- differences in amount of AER signal (Fgf)
Surface dwelling fish populations eyes vs. cave dwelling fish populations eyes
In surface fish they have expression of Pax6 and Shh
In cave fish Pax6 is not present and they have more expression of Shh than surface fish
Example of heterometry since there is a change in the amount of gene expression
Why would cavefish benefit from loss of eyes?
Cavefish have bigger jaws and more taste buds than surface fish
Therefore the hypothesis is the loss of eyes enables more room for a bigger jaw and tastebuds which would be beneficial in the dark
Solute size/number is determined by
- Rate of clock oscillations (formation time)
- Rate of axis elongation (PSM cell generation time)
Snake somites compared to other animals
More frequent notch clock oscillations ~~> difference in timing of notch clock/notch target expression. Hererochrony
Higher levels? Heterometry
Location being changed? Heterotopy
Loss of snake limbs in python
Embryonic pythons make a bind limb bud
Weak Shh expression results in failure to maintain AER and thus tiny “vestigial” hind limb forms
How does a limb keep growing?
Shh expression ultimately sustains FGF8, which keeps limb growth
FGF8 sustains Shh to maintain A/P patterning
Hox genes turn on ZRS enhancers which promotes Shh expression in ZPA
Why do pythons get tiny “vestigial” hind limb formation?
17bp snake specific deletion in ZRS therefore there is less Shh expression in the limb bud
Heterometry- change in the amount of Shh in limb bud
Loss of ZRS enhancer function = loss of python limb experiment
Used CRiSPR to delete 17bp from python to mouse. Resulted in serpentized limbs
Reduction = heterometry from the change in Shh expression in limb bud
ZRS enhancer function
ZRS sequences are highly “conserved” across limb-bearing vertebrates
Inserting other ZRS sequences (e.g. human, fish) into mice results in normal Shh expression and limb formation
Only time there is no Shh expression is when cobra, python or the ZRS gene is removed from the mouse
Teosinte to corn/maize
Could be considered alteration of Tga1 function which would be heterotypy
Glume is the protective fruit case on teosinte
Tga1 (teosinte glume architecture) is a txn. Factor important during maize war development
Tga1 mutation present in maize causes 1 amino acid substitution (K—>N) that results in more rapid Tga1 protein degradation —> as a result, glume does not fully form on corn/maize as it does on teosinte
