stem cells, maternal effect and genetic control of drosophila development

Question 1

pattern formation requires four things, what are they?

Answer 1

Cell proliferation

Cell specialisation - essentially how certain genes switch on/off, the gene expression of a cell

Cell interaction - Specialisation often requires interaction between cells

Cell movement (in animals, not in plants)

Question 2

different cell types arise by A____?

what are the two types?

Answer 2

asymmetric division

Intrinsic asymmetry - Prior to cell division, a cell fate determinant (CFD) becomes polarised in the cell, resulting in the CFD ending up in only one of the daughter cells, and instructs it to develop differently

Extrinsic asymmetry - cell division is normal, there is no cell fate determinant segregated. The daughter cells are in a slightly different environment, interact with different cells/experience different signalling from surrounding cells etc… external factors cause one of the cells to develop differently

Question 3

explain how lateral inhibition occurs

include why it shows P_F_

Answer 3

Two cells, contain factor X
X signals to prevent other cells from producing ‘X’
Two cells next to each other, both producing X, both trying to tell the other cell to stop producing X
A random? transient bias occurs, one cell has slightly more X, reducing X production in neighbouring cell

shows Positive Feedback -
s in one cell starts producing less X, the inhibition gets stronger as the difference in X between cells increases (‘amplifies asymmetry’)

Question 4

how can complexity/diversity be created very quickly with short range cell-to-cell signalling?

Answer 4

lets say a stem cell, A, is located next to another cell, B

A divides, and has two daughter cells in slightly different environments because one of them is now next to B, and is influenced to become ‘C’

C is now in a unique environment in that is is next to A and B, AND it acts as a new factor in the environment of A and B etc…

Question 5

what is a morphogen? generally how do they work?

Answer 5

signalling molecule that acts directly on cells to produce specific cellular response depending on its local concentration

A source cell produces the morphogen → diffuses → the surrounding cells have different concentration ‘thresholds’ resulting in different responses/fates in the cells

Remember - the morphogen in this scenario will be external, so a receptor is required on target cells for it to have an effect

Question 6

how can a morphogen cause different cell fates when it is uniformly distributed?

why can this be useful?

Answer 6

an inhibitor of the morphogen is distributed in a gradient instead
Morphogen activity is greatest where the inhibitor is at its lowest concentration (and vice-versa)
What’s this for - adds more control points, not just relying on initial diffusion of your morphogen

Question 7

How is a morphogen ‘read’ to result in a different outcome in different cells?

Answer 7

lets say the morphogen in question is a transcription factor (just one option, they can be many things)

in the promotor regions of the genes - lets say this TF targets three genes - there are often multiple binding sights for the TF, with different affinities

High affinity binding sites - don’t need a high conc. to bind - same ‘binding’ of TFs throughout the morphogen gradient
in the middle - slight tail off of response/binding as conc. of the TF decreases…
low affinity binding sites - means only binds at high concentrations

The concentration of these factors and how they interact with the target gene determine expression level

Overall expression level of a gene is affected by:
The ratio of of different TFs involved
Some TFs act as repressors, so ratio of initiators:repressors
TF binding sites in regulatory regions - different number of binding sites for different TFs and at different affinities

Question 8

why are drosophila good for studying development?

Answer 8

Segmented late stage larvae - maps perfectly to adult fly

So you’d know which segments should develop legs, wings etc…
Can investigate the genes that determine this

The clear mapping from larvae to adult, the obvious polarity = good model organism for development

good for mutagenesis screens - these need to be on large scale - hoping to hit as many genes as possible and observe outcome of losing/affecting their impact (so smaller organisms are good)

Question 9

what is a balancer chromosome and why are they used in screens?

Answer 9

has the full complement of the chromosome/all the genetic components, but has undergone rearrangement/its all mixed up. So the chromosome has the genetic components but cannot form crossovers - stabilises the mutation on a known chromosome, i.e. you can follow the specific chromosome from the male knowing it hasn’t changed

Question 10

why did Nüsslein-Volhard use a balancer chromosome?

Answer 10

allowed the researchers to maintain the mutagenised fly lines as heterozygote stocks, without the annoying problem of having to identify new heterozygotes after each mating - (basic genetics see a recessive gene inherited 1 Wt: 2 hets: 1 homozygote, so using a conventional strategy, you would always have a mix of WT and heterozygous flies in the next generation).

By using both the DTS-91 mutation and balancer, this allowed Nüsslein-Volhard and colleagues to ensure they generated the desired male and female genotypes by the third generation

Question 11

what cross did Nüsslein-Volhard do in their mutagenesis screen to identify genes controlling early stage development?

Answer 11

Males with a (recessive) phenotypic marker - in this case cinnabar mutation (white eyes, a recessive trait). Fed BMS (causes mutations, these mutations are what we want to study)

Bred with females with, on the same chromosome in question, a dominant temperature sensitive mutation (so when grown at high temperature the organism dies) and a balancer chromosome

Question 12

in Nusslein-Volhard’s experiment what was the F1 generation like?

F2?

Answer 12

looking at the males → high temperatures kill any males with the temp sensitive Chr, so all males have the balancer chr (and white eyes as they are heterozygous for cinnabar)
These are backcrossed with their mother

F2 -
high temperatures kill any offspring with temp. sensitive Chr, so all offspring have one of the mutagenised chromosomes from male right at the beginning, and one balancer chromosome (note - two balancer chromosomes = not viable so these die)
This is what you want - 1000s of individuals each with different stabilised mutations

Siblings from this cross are then crossed together, giving three possibilities (the main one were looking for is the homozygous for a mutation

Question 13

what were the three outcomes of the F3 generation in Nusslein-Volhard’s mutagenesis screen?

Answer 13

1. two balancer chromosomes = not viable
2. Heterozygous - one balancer and one mutant, not what were studying but useful in that these are same genotype as the desired flies from F2 so this line is maintained if needed
3. Homozygous for the mutagenised chromosome - the phenotype can be studied. If lethal you’ve got the heterozygous offspring ^ that should survive to be studied instead

Of the ones alive, its a 2:1 of heterozygous to homozygous mutants

Question 14

what are the four different kinds of segment identity genes (identified in the mutagenesis screen)?

Answer 14

Gap genes: involved in establishing large regions along the anterior-posterior axis of the embryo during early development

Pair-rule genes: define segmental boundaries, affecting alternate segments (mutants lack every other segment)

Segment polarity genes: involved in establishing polarity within each segment of the embryo (mutants appear as deletions, duplications or polarity reversals)

Homeotic genes: had previously been identified, roles in organ identity (more in later lectures)

Question 15

what was noticed about GAP genes?

Answer 15

expressed very early in development, just as transcription was being initiated in the zygote
Something must be initiating expression of the gap genes
Saw the unfertilised oocyte was polarised - something must be going on prior to fertilisation, something from the mother

Began to look for ‘maternal effect genes’

Question 16

how did Nüsslein-Volhard then go and look at maternal effect genes?

Answer 16

Gathered adult females with white eyes (new these were homozygous mutants from F3) and looked at their progeny

Question 17

what did Nüsslein-Volhard find when they looked at the progeny of F3 females?

Answer 17

WT larvae = had polarity, anterior terminal acron followed by the head, thorax, abdomen and finally the post. terminal telson

Mutants -
Deletion of terminal regions (acron and telson)
Deletion of posterior - specifically the posterior abdominal segment
Deletion of anterior regions (head and thorax)

Bicoid - mutants show an anterior deletion
Nanos - mutants show posterior deletion

Note - similar effect to but not the same as gap genes/ gap mutants

Question 18

axis formation is determined before fertilisation. How/what is needed?

Answer 18

In the egg chamber, Bicoid mRNA is tethered to anterior pole (surrounded by nurse cells)
At posterior pole, nanos mRNA was tethered

Other factors -
Need microtubule network for the positioning of BICOID and NANOS at either pole
Need a protein called gurken that comes before Bicoid and nanos

Question 19

what happens to Bicoid and Nanos after fertilisation?

Answer 19

Nucleotide probes used to detect mRNA for bicoid and nanos
Both were still tethered to their respective poles

Translation of the mRNA occurs

Antibodies for the proteins were used to identify their location
They each form a gradient across, highest concentrations at their respective poles

Question 20

what experiment was done to be sure that Bicoid is the anterior determinant?

Answer 20

Injected bicoid mRNA into mutant embryos
When doing this in anterior pole - got normal WT phenotype
When they injected bicoid into the centre of the body - embryo had the head in the middle with thoracic segments either side - mirror image duplication

Injecting bicoid into posterior pole - bicoid is present at both poles, each pole develops as the ‘anterior’, so two heads one at either end
So yes - bicoid is an the anterior determinant

Question 21

what are Hunchback and Caudal - after looking at them via in situ hybridisation?

Answer 21

Two other maternal effects genes (synthesised in nurse cells, transported to oocyte)

In situ hybridisation revealed their mRNA showed no polarity, uniform distribution
(After fertilisation. When ME genes are translated) hunchback protein gradient maps to bicoids, and caudals map’s to nanos

Question 22

what are the Bicoid, Nanos, Hunchback and Caudal proteins?

Answer 22

Bicoid = (K50) homeodomain transcription factor

Nanos = RNA binding protein

Hunchback = Zn finger transcription factor

Caudal = homeodomain transcription factor

Question 23

what relationship do Bicoid, Nanos, Hunchback and Caudal have?

Answer 23

Bicoid and nanos regulate hunchback and caudal levels -

Bicoid inhibits translation of caudal - hence caudal is found in high conc. At posterior pole, not the anterior pole

Nanos inhibits translation of hunchback RNA

Question 24

how does Bicoid regulate caudal?

Answer 24

TFs bind to DNA to regulate transcription.

BUT, bicoid also binds to the mRNA of caudal, on a binding site at the 3’ UTR of caudal transcript (a rare ability for a TF)

Interacts with an elongation factor that displaces the normal cap proteins of the mRNA, and in doing so blocks the ribosome from being recruited

Question 25

what single amino acid in bicoid is so important and why?

Answer 25

a single Aa in the K50 homeodomain - Arg 54

is key to bicoid’s binding to DNA and to RNA - uses the same domain to bind to both

If mutated it cannot bind to and inhibit the caudal transcript (but can still bind to DNA for transcriptional activity)

Question 26

aside from inhibiting translation of Caudal, what does Bicoid do?

classify Hunchback

Answer 26

regulates expression of Hunchback, reinforcing its gradient, and regulates expression of the next step down in the hierarchy - GAP genes

maternal effect gene because its expressed pre-fertilisation, but also a GAP gene as it’s expression is regulated by Bicoid

Question 27

explain what is meant by a drosophila embryo being called a ‘syncytium’

Answer 27

In the fertilised egg, it’s rapidly dividing nuclei, no cell membranes separating them, so the nuclei are directly exposed to the M-E proteins
The nuclear division occurring is rapid

It is the rapid development at this stage that first made Nüsslein-Volhard reassess her mutant population for maternal effect mutants (as well as the observed polarity prior to fertilisation I think)

Eventually nuclei migrate to periphery for cellularization - formation of cells/boundaries

Question 28

maternal effect genes are the earliest to be expressed - then what?

Answer 28

then GAP genes (also early, clear as both mutants of these genes had large deletions)
these are regulated by maternal effect genes

You’ve also got pair-rule genes, segment polarity genes and homeotic genes

Question 29

what are parasegments?

Answer 29

don’t fully align to physical segments
Align to gene expression domains instead. Often map better to regions deleted in mutations

Question 30

how are GAP genes designed for regulation by Bicoid?

Answer 30

GAP genes expressed at the anterior of the embryo (still a syncytium) were found to have binding sites for Bicoid in their promoter regions

These binding sites had different affinities for Bicoid - the affinity of the promoter regions of these GAP genes for Bicoid plays a major role in defining where these genes are expressed (expression domain)

Question 31

while Hunchback is still localised to the anterior, why is it’s expression domain quite wide?

do you get any Hunchback at the posterior?

Answer 31

HUNCHBACK promoter has multiple Bicoid binding sites, some with high affinity and some with low affinity

While still localised to the anterior, high affinity sites lead to wider expression domain , across the whole anterior

some expression at the posterior, but this is due to another GAP gene ‘TAILLESS’

Question 33

where is the GAP gene orthodenticle expressed?

Answer 33

only expressed in a very tight region at the anterior (where bicoid is in high conc), because it’s promoter has (3) low affinity Bicoid binding sites - so Bicoid only binds when in high concentrations

Question 34

explain what is meant when the regulation of GAP genes is called ‘dynamic’, and include an example

Answer 34

yes, BICOID (and then Hunchback) are essential in establishing early expression patterns/areas of the GAP genes, Bcd regulates a lot of the gap genes

but the regulation is ‘dynamic’ - GAP genes, once expressed, regulate each other (mostly inhibition of one another but not always)
This gives stable, tight expression zones/domains

E.g. Kruppel and giant inhibit each other’s expressions so that they aren’t expressed in the same place

Question 35

pair-rule genes - when are they expressed, what are their expression domains like?

Answer 35

Expression of pair rule genes starts when nuclei move to and cells begin to form at the periphery (after syncytium has undergone 13 divisions)

Expression domains = a bit fuzzy at first but by 3.5 hrs post fertilisation the domains are clear strips across the embryo

Question 36

there are two categories of pair-rule genes, what are they?

Answer 36

Two categories - primary and secondary.

Three primary pair-rule genes, HAIRY, EVEN-SKIPPED (EVE) AND RUNT. primary means expressed first,

they are regulated by the genes above them in the hierarchy, ME and GAP genes

Question 37

describe what the sequence of the EVE gene is like, and how this is important in determining expression domain

Answer 37

In Situ hybridisation and sequencing of the gene showed -

Very large gene (20Kb long)
The regulatory region is upstream AND downstream of the coding sequence

Regulatory region has different sections/modules called stripe #_, each of these ‘modules’ determines expression in a specific stripe…
How - each section has different binding sites for maternal effect and GAP proteins

Question 38

how was the whole stripe #_ thing shown for pair-rule genes regulatory regions?

Answer 38

Removed the stripe #2 enhancer module and put it in front of a reporter gene to see where it’s expressed
(so Eve was no longer expressed in stripe 2 but the reporter gene was)

Question 39

explain how EVE’s stripe 2 domain is determined

Answer 39

Bicoid and Hunchback are positive regulators of EVE, binding to the stripe 2 module

Giant and Kruppel are negative regulators/inhibit EVE expression at Stripe 2 module

stripe 2 enhancer is not active at high BICOID concentrations right at the very anterior (odd)
but is very active a little further down…

Bicoid may have dropped off, but Hunchback is still in high conc., so is acting as a positive regulator…

high BICOID corresponds with high GIANT - the reason expression levels are confined is due to the negative regulators GIANT and KRUPPEL defining both the anterior and posterior boundaries of stripe 2

NOTE - there are also binding sites for caudal, knirps and tailless, so its complicated but you get the picture

Question 40

secondary pair-rule genes - explain using the example of EVE and fushi tarazu

Answer 40

Again, genes above in the hierarchy establish initial expression domains, but this is further defined by regulation within the category (pair-rule genes regulating pair-rule genes)

EVE (primary) regulates expression of fushi tarazu (secondary)

Once EVE is translated and expressed in its correct regions, it then inhibits expression of ftz, so ftz is only found where EVE is not (alternate segments)

Question 41

the embryo is now in the blastoderm stage - this means…?

Answer 41

cell membranes formed mean cell-to-cell signalling is required/no free diffusion
Cell fate begins to be determined

Question 42

segment polarity genes are more involved in regulating C_F_?

Answer 42

More involved in regulating cell fate, have homologs in higher eukaryotes like mice and humans

Question 44

three segment polarity genes involved in hair - what are they and tell me more about the hair?

Answer 44

Engrailed, wingless and hedgehog
Normally, each thoracic and abdominal segment has SECTIONS of hair-like structures on the dorsal cuticle -

1/primary cell row (acc. in the overlapping parasegment) has denticles (large spikes)

2 row is smooth

3 - next two rows - small thick hairs

4 = several rows of fines hairs

This organisation is disrupted in mutants of the segment polarity genes WINGLESS, HEDGEHOG and ENGRAILED (no smooth cells, hair all over and all the same)

Question 45

explain how engrailed (En), Hedgehog (HH), Wingless (Wg), EVE and FTz interact

Answer 45

EVE and Ftz positively regulate En
So En is always expressed in the last/posterior cell rows of a segment

Engrailed is a TF
+vely regulates HH
So cells expressing En also express HH

HH is secreted
Diffuses to surrounding cells, causes signalling cascade to express Wg

EVE and FTz repress Wg
So HH may promote Wg, but it can only be expressed in the cell between EVE and Ftz segments

Wg is secreted
And positively reinforces expression of engrailed

Result:
In each segment, last few posterior rows of cells express engrailed and therefore HH too…
Cells immediately anterior express wingless (as a result of HH, only that one row of cells, not other surrounding cells because of the repression of Wg by both EVE and Ftz)

This positive interaction leads to a stabilisation of HH and WG expression and stabilises their expression boundaries in the observed cells