Developmental Genetics (15-23)

What sort of symetry do organisms show?

• Bilateral symmetry - Human, shark, fly

• Radial symmetry - Arabidopsis flower

Question is, what genes regulate these processes

What are the cell processes that occur for a single cell to develop complexity (grow into a whole organism)?

• Cell proliferation

• Cell specialization

• Cell interaction

• Cell movement (not in plants)

What is Intrinsic Asymmetric Division?

• Where a component becomes polarised in the mother cell prior to and upon divison

• The unequal distribution of this component leads to daughter cells with different fates

What is Extrinsic Asymmetric Divison?

• Daughter cells are identical upon divison however signaling external to the cell generates a different fate

Where is asymmetric divison often found?

• Right at the begining of embryo development when the fertilised egg divides to give daughets with different fates

• Also found in association with Stem Cells, where division generates a daughter with stem cell activity and a seccond with a new fate

What is Lateral Inhibition in Extrinsinc Asymmetry?

Where two daughter cells excrete a factor ‘x’ that will inhibit the others production of that factor ‘x’

Any difference in the concentration of ‘x’ will lead to asymmetry

Bassicaly a fight where only one can win

How can close range cell to cell signaling lead to increasing complexity?

Signals from one daughter cell (a) can induce division in the other(b) to result in a new cell type(c), this cell can then induce divison from the two original cells to result in two more new cell types (d&e)

Quickly generates increased complexity

(Cell autonomous divisions do occur still, dividing a certain number of times before differentiating)

What is a morphogen?

A signalling molecule that acts directly on cells to produce a specific cellular responses depending on its local concentration. Control gene expression in a spatialy dependant manor

What are threshhold levels within a morphogen gradient?

Different threshold levels occur down a morphogen gradient, causing cells to aquire different fates from the same morphogen depending on conc of morphogen

What is an example of a radial morphogen gradiant?

Butterfly spots

What can inhibitors do to a morphogen gradient?

Morphogen inhibitors also can have a gradient, this negatively regulates morphogen activity across a gradient.

What is a benefit to inhibition gradients alongside morphogen gradients?

Allows for a greater level of control than purely relying on morphogen diffusion

How does a morphogen gradient translate to differences in cell fate?

If the morphogen is a transcription factor (TF) (for example), promoter regions for different genes can have different affinitys for the TF. This means that a promoter region with a low affinity for the TF will only be switched on where the TF is in high conc (higher up the gradient)

What do Repressor TF factors mean for gene regulation?

Make it more complicated, introduce a greater level of control

You can have scenarios where both activator and repressor factors are pressent and whether the gene is expressed or not is dependant on the ratio

What reason was Drosophila adopted as a model organism for early development?

The adult body plan can easily be mapped to a series of segments in larvae, from a patterning perspective there is obvious polarity

How did Nusslein-Volhard and colleagues use mutagenesis and balancer chromosomes in Drosophila experiments?

They mutagenized male Drosophila and mated them with females carrying balancer chromosomes. Single-male descent produced inbred lines with stable mutations. Mating siblings allowed them to study homozygous stocks without re-identification each generation, ans the DTS-91 mutation ensured corect genotypes by the third generation

In Drosophila, is axis formation determined before or after fertilisation?

Before

Is NANOS mRNA tethered at the posterior or anterior of the Unfertalised Oocyte?

Posterior

Is BICOID mRNA tethered at the posterior or anterior of the Unfertalised Oocyte?

Anterioir

What happens when a BICOID mutant is injected with anterioir cytoplasm from wild type eggs?

The mutant is rescued as polarity is determined and growth continues as normal

What does the position of BICOID mRNA determine?

Head position (they made flys with 2 heads)

What happens to BICOID and NANOS mRNA upon egg fertilisation?

The tethered mRNA is translated and the proteins form concentration gradients along the axis of the egg

What is essential for targeting of NANOS and BICOID mRNA to anterior and posterior poles?

Polarised microtubule network

What is different about the distribution CAUDAL and HUNCHBACK mRNA compared to BICOID and NANOS mRNA?

CAUDAL and HUNCHBACK mRNA is distributed evenly throughout oocyte/early embryo

Where is HUNCHBACK protein found?

Anterior pole

Where is CAUDAL protein found?

Posterior pole

What is different about NANOS, compared to BICOID, HUNCHBACK and CAUDAL proteins?

• BICOID, HUNCHBACK and CAUDAL are all transcription factors capable of activating or repressing a gene

• NANOS is a RNA binding protein

What does BICOID protein bind to and what does it inhibit?

Binds to CAUDAL mRNA and inhibits its translation (preventing CAUDAL proteins)

What does transcribed NANOS bind to and what does it inhibit?

Binds to HUNCHBACK mRNA and inhibits its translation (preventing HUNCHBACK proteins)

What is unusual about BICOID, what does it bind to?

• It has dual functionallity

• It binds to:

  • DNA (promoting CAUDAL mRNA translation)

  • CAUDAL mRNA (ensuring translation and preventing degredation

What is a syncytium, what happens in a drosophila syncytium?

• A mulinucleate structure without cellular boundries

• The drosophila syncytium is where the maternal effect gradients work

What are the 3 types of segment identity genes in drosophila?

• GAP genes

• Pair-rule genes

• Segment polarity genes

What do the GAP genes define?

Broad regions of the embryo (head,thorax,abdomen), mutations result in large deletions of the body plan

What do the Pair-rule genes define?

Alternate segments, such that mutants lack every other segment

What do segment polarity genes define?

Define the adult segment plan, mutants show defects in every segments

What is the Hierachical regulation of segmentation is drosophila, and what is it initiated by?

1. GAP genes, broad regions

2. Pair-rule genes, alternating segments

3. Segment polarity genes, define adult segment plan

Initiated by maternal effect genes

Which GAP gene mutants lack central segments of the embryo?

Kruppel mutants

How is the BICOID protein gradient decoded by promoter elements?

• Promoter elements have different binding affinities for BICOID

• This means that those with higher affinities will bind across a much broader area as they can bind where BICOID conc is low still

• Promoter elements with low affinities for BICOID will show tight expression as they will only bind where BICOID conc is high

What affinities are the BICOID binding sites on HUNCHBACK and what is the expression as a result of this?

2 high affinity for BICOID and 1 low

• Resulting in a broader expression of HUNCHBACK

What affinities are the BICOID binding sites on ORTHODENTICLE and what is the expression as a result of this?

3, low affinity for BICOID

• resulting in a tight expression (Small area) in the anterior domain

How do GAP genes result in destinct expression zones in different areas of the embryo?

GAP genes regulate each other, mostly by inhibiting expression of other GAP genes

• Resulting in tight/stable expression patterns

• Some are regulated by multiple other GAP genes

When does pair-rule gene expression occur?

After the syncyntium has undergone 13 nuclear division cycles and cells begins to form at the periphery of the embryo

How are pair-rule genes expressed, and what are the 3 primary pair-rule genes?

Initially fuzzy, but rapidly becomes distinc as stips across the embryo

1. HAIRY (H)

2. EVEN-SKIPPED (EVE)

3. RUNT (RUN)

How big is the EVE regulatory region?

~20kB

What do the modules on the EVE gene contain binding sites for?

Maternal effect proteins and GAP proteins

How can you demonstrate that an indavidual EVE enhancer module determines stripe specific expression?

By cloning an indavidual stripe enhancer in front of a LacZ (or other reporter) gene. (then visualise by staining with X-GAL)

What are the 2 positive regulators of Stripe 2 EVE expression?

• BICOID

• HUNCHBACK

What 2 proteins inhibit the stripe 2 module of EVE?

• GIANT

• KRUPPEL

What is the result of the positive regulators and inhibitors on the stripe2 mosule of EVE?

A tight band of expression where threre is no KRUPELL or GIANT, but threre is BICOID and HUNCHBACK so the gene is actually expressed

What is primary pair-rule gene expression determined by?

Maternal effect and GAP

What is needed by secondary pair-rule genes to determine expression, and what is an example of a secondary pair-rule gene?

Primary pair-rule genes

Fushi Tarazu (ftz)

What effect does EVE have on FUSHI TARAZU?

FUSHI TARAZU is broadly expressed throught the whole embryo. After cell cycle 13, EVE is expressed and acts as a repressor on FUSHI TARAZU, casing an alternating striped expression pattern of FUSHI TARAZU

Where is ENGRAILD (en) expressed?

In cells that express Ftz but not Eve, at the anterior edge of each parasegment

What regulates engrailed (en) expression?

Activated by FTz and repressed by Eve

What gene is expressed as the same cells and Engrailed?

Hedgehog (hh) - direclty activated by En

What is the function of the Hedgehog protein?

It activates wingless (wg) expression in adjacent anterior cells

How does Wingless signaling affect engrailed expression?

Wingless maintains engrailed expression in adjacent posterior cells - forming a feedback loop

What is the result of the En-Hh and Wg signaling loop?

It maintains stable segment boundaries and establishes cell polarity in each segment

What Chromosome are the homeotic genes on and how many of them are there in Drosophila?

• Chromosome 3

• 8 genes

What are the two complexes called that the Homoetic genes of drosphila are organized into?

• Antennapedia complex

• Bithorax complex

What is signifiacnt about the sequence of homeotic genes in drosophila?

The sequence of the genes in both Antennapedia and Bithorax complexs correspond to the order in which they are expressed along the body axis

This organisation is conserved in higher vertebrates such as humans and mouses (squeak)

How are Homeotic genes regulated by GAP and pair rule genes?

• Expressed based on promoters with high and low affinity binding sites

• They are also regulated by other homeotic genes repressing each other

What is epistasis?

Where alleles of one gene mask or overides the effects of another

What is posterior prevelance?

When homeotic genes in drosophila are epistatic to their anterior neighbour

This means if both genes are expressed in a parasegment then the posterior genes identity wins

What happens when you use a heat shock mutant to turn on Ubx (homeotic gene) everywhere?

All parasegments anterior to Ubx’s normal domain domain will now express Ubx (posterior prevelance)

What is the phenotype of an Antennapedia mutant?

Legs appear instead of antenna

What does Antp block the expression of in leg segments of drosophila?

Blocks the expression of Homothora and Extradenticle, leading to leg growth

What happens becasue of Antp being ecotypically switched off in the head region of drosophila?

Homothorax (hth) is switched on, meaning extradenticle(Exd) expression is also switched on resulting in Antena development

What actualy do homeotic genes code for?

Homeodomain trascription factors

• 60 amino acid DNA(and RNA e.g. BICOID) binding domain

• Regulate genes that specify tissue/organ primordia

Not all homeodomain genes are homeotic genes, not all homeotic genes contain a homeodomain

What is the role of Polycomb and Extra sex combs?

They are responsible for the methylation of histone complexes that condence the chromatin making transcriptional machinery inaccesible and silencing the genes.

This offers a mechanism by which homeotic genes can be silenced in specific regions once GAP and Pair-rule genes are turned off

Ensure homeotic pattern is maintained and stable in adult fly

What happend to GAP and pair-rule genes after a while?

They are only around fro develompent, they are not expressed for the lifetime of the drosophila, they show temporal control

What happens in a polycomb mutant?

Expansion of the expression domain, when GAP and pair-rule genes not there to define region of homeotic gene. Polycomb would normally lock the pattern, through cromatin regulation.

What do Trithorax genes do?

• Maintain gene activity

• Antagonise polycomb gene

• Maintain correct homoetic expression domains

What is the ultimate output of the Wingless (Wnt) signalling pathway?

Gene regulator beta-catenin is activated and Wnt target genes expressed (on)

What is in the complex that degrades beta-catenein when Wnt is abcent?

Scaffold proteins

• APC

• Axin

Kinases

• Caesin kinase 1

• Glycogen synthase kinase 3

What binds the Axin/APC complex when Wnt signal is present?

Disheveld, inactivates axin/APC complex preventing it from degrading beta-catenin wich can bind Wnt target and activate DNA

What is the pathway when Wnt is pressent and bound to Fizzled?

• Both Frizzled and LPR activated by bound Wnt

• Disheveld activated which binds the Axin/APC complex

• Axin/APC complex prevented from degrading beta-catenin as Kinases not bound (GSK3 & CK1)

• beta-catenin free to bind Wnt target genes and activate transcription (Wnt genes ON)

What is the pathway when Wnt is NOT bound (pressent)?

• LRP and Frizzeld inactive

• Disheveld inactive

• This means Axin/APC complex is active and the kinases(GSK3 & CK1) are free to degrade beta-catenin

• Genes remain OFF as beta-catenin cannot bind

What can a APC mutation cause?

Colorectal Cancer

• Polyposis Colon is a colon covered in hunderds of polyps as a result of APC mutation

• There is a high chance that one of these will become malignant

• This is caused by the over activation of beta-catenin (Wnt gene activation) as APC is not there to downregulate it

What happens in mice where Wnt4 is targeted for a knockout?

Wingless pathway is blocked → incorect activation of gene targets

Leads to incorrect kindey and gonad development → ovaries start to syntheise testostorone and therefore take on male charecteristics

What is the pathway when Hedgehog binds?

• Hedgehog binds activating patched protein

• Smoothened (signal trasducer) inhibits Ci degredation by binding PKA and slimb kinases

• Ci binds to Hedgehog responsive sites on DNA activating genes

What is the pathway when Hedgehog is not present?

• Patched protein is inactive

• Smoothend signal transucer is inactive, meaning PKA and Slimb kinases are free to cleave Ci

• The cleaved Ci binds to the HH target DNA but acts as a repressor

What are the 3 Hedgehog genes found in vertebrates and what do they do?

• Sonic Hedehog - Limb development, Neural differentiation, Facial morphogenesis

• Desert Hedgehog - Spermatogenesis

• Indian Hedehog - Bone growth

What happens when Shh (sonic hedgehog) in posterior region of wing bud is transplanted to the anterior region (whilst leaving posteroir pole in tact)?

You get a mirror-image duplication of the digit pattern in the wing

What does the Shh transplant in wing buds demonstrate about sonic hedegog?

Indicates morphogen like behavoir, its concentration gradient affecting decisions (also found in mouse limb development)

What was the findindgs from the Sonic the hedgehog digit experiments in mice, (Gli3 & Gil3R, Ci ortholouges)?

• The ratio of Gli3 and Gli3R(epressor) determines digit identity and number

• It is belived that it is a gradient of Shh which results in changes in the ratio of the active ans repressive Gli3 forms

What is the effect of loss of Shh in Ventral Induction?

• Forebrain can fail to divide

• Shh expressed at the ventral centre and structures derived from here can be abscent

• Defects in Shh can lead to mild to severe Holoprosencephaly, most common brain defect in humans - lethal

• Can also lead to an undivided eye, Cyclopia

Why can grazers that eat California corn lily have cyclopia?

The plant produces a steroidal alkaloid called cyclopamine, which binds directly to the smoothened and prevents activation of the hedgehog pathway, even in presence of Hedgehog binding to patched

What is the link between hedgehog signaling and cancer?

• It was found that patched is a tummor supressor gene associated with gorlin syndrome - basal cell carcinoma (skin cancer)

• Mutations in patched may lead to constitutive activation of Gli targets (Ci ortholouges)

• Autosomal dominant so only one copy of defective patched gene increases the risk of developing symptoms

What is one of the treatments of cancers associated with Hedgehog signaling?

• Giving cyclopamine or cyclopamine analouges, as they inhibit the overactive HH pathway

What does the HomC complx in drosophila and the Hox complex in humans and mouse have in similar?

Theyre homolouges and show anterior-posterior expression

Why is it not so easy to answer the question of wether Hox expression domain correlates with vertebrate type in humans and mosue?

Humans and mouse have redundancy, you can’t knockout a single gene as hoxc13 and hoxd13 will counter the loss of hoxa13

How were Hox gene knockouts created in mice given their redundancy and what was the result?

They generated knockouts in single genes and then cross the mice together until they eventially had tripple knockout genes in mice

• A tripple Hox10 mutant - Thoratic replace lumbar vertebrae

• A tripple Hox11 mutant - Lumbar replace sacral vertebrae

What ruel was found to be followed in mice as in drosophila?

Posterior superiority, Posterior genes repress the activity of those anterior to them, When all Hox 10 genes knocked out, posterior expression of hox 9 expande where hox 10 should be as it is not there to repress it

What is the Human ortholouge of Posterior sex combs (PSC) and what role does it play in cancer?

• BMI1

is an oncogene with a role in several types of blood cancers. The function of BMI1 has been linked to its role as a repressor of the cell cycle regulator CDKN2A, this functions to inhibit cell cycle G1 progression. In B lymphomas, BMI1 is overexpressed and thi sleads to the inhibition of CDKN2A function and unregulated cell growth.

What is the plant version of a stem cell?

Meristem

What polarity and symetry is seen in the plant cell embryo at the heart stage?

• Apical-basal polarity

• Bilateral symetry

What are the 4 concentric whorls in the Arabidopsis flower?

• Whorl 1 - Sepals (outermost)

• Whorl 2 - Petals

• Whorl 3 - Stamen

• Whorl 4 - Carpels (innermost)

What makes a mutant homeotic?

When its deletion results in a replacemet of that section, not deleted entirely

What were the 3 homeotic mutants discovered in arabidopsis and what were the phenotypes?

• Apetala1 - Only carpels and stamen

• Pistillata - Only sepals and carpells

• Agamous - Only sepals and petals