homeotic genes and drosophila to vertebrates

Question 1

what do homeotic genes do?

what is the ultrabithorax mutant?

Answer 1

Homeotic genes regulate organ identity on an adult fly
These mutants were amongst first to be identified in drosophila, namely the ultrabithorax mutant

Homeotic mutants cause a replacement of one part of the body plan with another

Ultrabithorax mutant - two sets of wings.
T2 = where wings are expressed. When Ubx is mutated, T3 appears to be replaced with another T2 segment, causing two sets of wings

Question 2

what did E.B. Lewis say about homeotic genes?

Answer 2

something like if you have an understanding of these homeotic genes you can start to understand where certain body plans come from, including in terms of evolution

Question 3

homeotic genes - where are they found, what is their organisation like?

Answer 3

Found on Chr 3

Separated into two complexes -
antennapedia complex and bithorax complex

Key - the sequence in which these homeotic genes appear in these complexes on the Chromosome = the order of expression along the ant/post axis
This idea is conserved in (most) higher organisms

Question 4

homeotic genes code for what?

Answer 4

in drosophila and in higher invertebrates, they are mostly homeodomain transcription factors

BUT NOTE - not all homeodomain transcription factors are homeotic genes (e.g. bicoid), and not all homeotic genes have homeodomains

Question 5

briefly, how are homeotic genes regulated?

Answer 5

GAP and pair rule genes (step up in the hierarchy) determine where homeotic genes are expressed

Homeotic genes regulate each other’s expression, via epistasis (or posterior prevalence)

Question 6

explain what is meant by epistasis, or posterior prevalence, when referring to homeotic genes, using the ultrabithorax mutant as an example

Answer 6

Epistasis -
A homeotic gene’s effects are masked in the regions posterior to its own, by the homeotic genes in those regions that come after on the Chr/complexes

Ultrabithorax example:
Antp is expressed in T2, where wings normally are. Ubx normally masks Antp expression in the region posterior to this, T3
This is why in Ubx mutants, there is expanded Antp expression, and T3 segment is now like another T2 segment with a second set of wings

Question 7

describe the experiment that showed homeotic genes work by epistasis

Answer 7

Antp - is expressed in parasegment 4 overlapping with T2
Ubx - is expressed in parasegment 6 overlapping with T3

Heat shock promoter was placed in front of the Antp gene
Heat shock applied and Antp was uncoupled from it’s regulatory domain through which it’s expression is normally controlled by GAP and pair rule genes

Result = Antp was expressed across the body, all regions anterior to parasegment 4 were converted to P4 also, but the posterior regions were not affected because the homeotic genes there still mask Antp effects

same thing was done for Ubx, same results seen

Question 8

antennapedia mutant -

explain how legs vs antenna develop in WT, then what is seen in Antp mutants

Answer 8

WT, Where legs should be - the leg primordium:
Antp is ON/expressed
Antp inhibits hth (homothorax), so Exd (extradenticle) remains in the cytoplasm and legs develop (kind of like a default?)

WT, Where antenna should be -
(antennal primordium) Antp is OFF (not expressed)
This means it cannot suppress hth so its ON, and can allow Exd to move from the cytoplasm to the nucleus, resulting in antenna development instead of leg development

Mutant - Antp IS expressed in antennal primordium and inhibits hth, this ‘switches off’ antenna sulphate, you then get leg development (default)

Question 9

aside from spatial control of gene expression, what else is seen?

Answer 9

expression also changes over time

GAP and pair rule genes are eventually no longer expressed
How then are homeotic genes prevented from suddenly being expressed in the wrong places…

by temporal control

Question 10

how is the pattern seen in larvae ‘locked’ when GAP and pair rule genes are no longer expressed (and aren’t around to control homeotic genes)?

Answer 10

Polycomb protein is part of the multi-protein complex ‘Polycomb Repressive Complex1
extra sex combs - part of Polycomb Repressive Complex 2

These complexes control Chr structure - when GAP and pair rule genes are turned off:
these complexes are guided to Polycomb response elements
Bind and condense chromatin

Cause Inaccessibility to transcription factors and machinery
Prevents homeotic gene expression now that GAP and pair rule genes aren’t on to regulate them

Question 11

how were the polycomb repressive complexes found? what is seen in mutants?

Answer 11

Looked at a mutant of ‘extra sex combs’ (part of one of the complexes) where all the segments became abdominal segments

Used an antibody to find where a protein called ‘polycomb’ bound across the drosophila genome

Turns out it bound to both antennapedia and bithorax complexes (the homeotic genes)

If Polycomb genes mutated - GAP and pair rule genes off, yet homeotic genes not turned off by PRCs so e.g. AbdB is expressed everywhere in the body and all segments become abdominal

Question 12

quick summary of polycomb genes?

Answer 12

responsible for gene silencing by histone modification

Pattern is maintained and stable in adult fly

Maintains correct homeotic expression domains

Question 13

what are the opposite of polycomb genes?

Answer 13

Trithorax genes

These are for maintaining expression, e.g. of homeotic genes in the correct domain when required

Summary -
Maintain gene activity

Antagonise Polycomb Genes

Maintains correct homeotic expression domains

Question 14

the hedgehog pathway - what happens in the absence of Hedgehog?

Answer 14

1. The receptor, Patched, is inhibiting the protein Smoothened
2. Ci is held in place by a complex in the cytoplasm
3. Ci will be phosphorylated and cleaved by a complex containing PKA and Slimb
4. Forming a version of Ci that moves to the nucleus to act as a repressor (Ci-rep), preventing transcription of HH targets

Question 15

Hedgehog pathway - what happens in the presence of Hedgehog?

Answer 15

1. HH (ligand) binds to receptor, Patched, inhibiting it

2 Patch no longer inhibits Smoothened (@ membrane)

3. Smoothened inhibits PKA and Slimb, preventing them from phosphorylating Ci (cubitus interruptus)
4. Ci can move to nucleus, acting as a Tf and activating transcription of HH targets

Question 16

in vertebrates, what are the different kinds of Hedgehog genes?

Answer 16

Sonic HH → limb development, neural differentiation, facial morphogenesis

Desert HH → spermatogenesis

Indian HH → bone growth

Question 17

in chicks, how was it shown the sonic hedgehog acts as a morphogen?

Answer 17

In-situ hybridisation was used on chick embryos to look at expression of sonic HH

Looking at the wing bud - there is a distinct sonic HH - expressing domain at the posterior

Taking one of these domains and adding it to the anterior of a wing bud, so that there is a high conc. of sonic HH at the anterior and posterior, resulted in a mirroring of digits(due to opposing gradients),

Suggesting digit formation and identity relies on a gradient of sonic HH as a morphogen

Question 18

mice digit identity - what 2 genes are involved and explain the phenotypes seen when either, or both are mutated

Answer 18

Genes involved:
Sonic HedgeHog (S-HH)

Gli3, a homologue of Ci, meaning it also has a repressor version when cleaved…GLi3R

No S-HH -
results in loss of digits, just one forms. If S-HH isn’t working, all Gli3 is cleaved (no Gli3 protection) to its repressive form, Gli3R, so none of the target genes are activated, they are instead all repressed

No Gli3 -
you don’t get distinct digit identity, you just get the default which is polydactyly (because no Gli3 means no Gli3R, so there are no gradients of sonic HH targets, just uniform expression)

no S-HH or Gli3 -
An example of epistasis. No S-HH or Gli3 - a double mutant. Phenotype is the same as above, where there is S-HH but no Gli3. meaning → Gli3 is required to see the outcome of the S-HH pathway

Question 19

in mice, what determines digit identity and number?

Answer 19

Gli3:Gli3R - this ratio determines digit identity and number.

No Gli3 = no ratio at all

Question 20

what does sonic HH do in the CNS?

Answer 20

HH seen in brain of developing embryo (in-situ hybridisation)
Essential for separation of hemispheres

Loss of Sonic HH pathway = defects in ventral induction, causes holoprosencephaly (hemispheres don’t separate), you get forebrain and facial development defects, including separation of the eye field so can result in cyclopathy

Question 21

what is cyclopamine? what does it do?

Answer 21

A steroidal alkaloid found in veratrum californicum (corn lily). Animals that graze on it can give birth to cyclopathy babies…

Cyclopamine inhibits smoothened = no protection of Ci, its all cleaved to its repressive form, gene targets of HH are not transcribed/pathway is inactivated

Question 22

how can cyclopamine be useful?

Answer 22

Patched is a tumour suppressor gene

Mutations cause a cancer called Gorlin syndrome
No patched = smoothened is never inhibited, so Ci is constantly protected from being cleaved, and is always in its active form, activating transcription of target genes too much

Treatment with cyclopamine can be used to inhibit smoothened (as mutated patched now cannot)

Question 23

wingless - what happens to the wingless pathway when there is no Wg present?

Answer 23

Dishevelled remains inactive

protein complex (Axin, APC, GSK3, CK1) free to degrade B-catenin

transcription of target genes NOT activated

Question 24

wingless patheway - what happens when Wg is present?

Answer 24

Binds and activates receptor complex, frizzled + LRP

Activates the cytoplasmic protein Dishevelled

Dsh phosphorylates protein complex (Axin, APC, GSK3, CK1) preventing it from degrading B-catenin

B-catenin (armadillo) stable + activates transcription of target genes

Question 25

wingless pathway - what is ectopic activation?

Answer 25

mutations of the proteins found in the protein complex would result in B-catenin not being degraded, pathway always active and genes being transcribed when they shouldn’t
Seen in colorectal cancer, results in polyps

Question 26

Wg4 vs its mutations?

Answer 26

found in kidneys and gonads, when knocked out you get halted kidney development and instead of female gonads you get testosterone production

Question 27

mice and human equivalent of homeotic genes?

they show R_?

Answer 27

In mice and humans there is conservation of the structure of HomC (the two complexes of homeotic genes on Chr3 in drosophila)

In mice (and human), the orthologues are Hox genes, found spread across a few chromosomes

E.g. mice have Hox complexes A - D, with A, B, C, and D each on different chromosomes

These Hox complexes show redundancy - e.g. there’s an A10, C10 and D10 which can control the same developmental processes, so to see what happens without that gene you’ve got to knock all the 10s out

Question 28

what happens when you knock out ALL the Hox10 genes? this shows that…?

Answer 28

associated with lumbar vertebrae
When all of the Hox10’s are mutated in mice, the lumbar vertebrae are replaced by thoracic vertebrae…

Hox genes in mice showed epistasis/posterior prevalence like in drosophila

Question 30

drosophila are an essential stepping stone in research on developmental genetics because?

Answer 30

most knowledge of the following pathways came initially from drosophila, and were then found (or homologues) in higher organisms/vertebrae