Lecture 1 - Segmentation in flies

Question 1

Why are developmental experiments often done on flies?

Answer 1

• many of the genes that control fly development are the same types of genes that control development of vertebraes
• sophisticated genetics
• already have a detailed understanding

Question 2

What is the structure of the anteropostior segmentation of the adult drosophila?

Answer 2

• at the anterior there are 3 head segments, 3 thoracic segments
• 8 abdominal segments at posterior

Question 3

What are the different life stages of Drosophila?

Answer 3

Hatches from egg as a lava

lava grows and later metamorphoses to form an adult fly

Question 4

What are the features of the drosophila larvae denticle belts?

Answer 4

• denticle belt of each segment has a specific pattern of bristles
• denticle belt sticks out the ventricle side
• noted by dissolution of larval cuticles by Nusslein-Volhard

Question 5

What was the process of genetic screen used by Nusslein-Volhard to look for developmental mutants?

Answer 5

Forward genetic screen (1980)

-make mutants and look at the phenotypes

Question 6

What are the three types segmentation genes in drosophila?

Answer 6

Classified by mutant phenotypes
Gap genes: lots of cuticles missing, big continuous gaps
Pair-rule genes: missing alternate segments (odd/even)
Segment polarity genes: segments were mirror image diplications of each other, as if the polarity of each segment was reversed

Question 7

What was the hierachy of segmentation genes identified by Nusslein-Volhard?

Answer 7

Maternal gradient
Gap genes
Pair rule genes
Segment polarity genes

Question 8

What is a multinucleatied syncital blastoderm and when is it present in Drosophila?

Answer 8

• Early drosophila embryos form a multinucleated syncitial blastoderm, nuclei with shared cytoplasm migrate to the edges and pole cells for at the posterior
• developmental patterning is set up in a syntium
• Synctium: when nuclei have a shared cytoplasm
• repeated rounds of nuclear dividion occur without cell division

Question 9

What occurs at the syncitial bastoderm stage in drosophila?

Answer 9

-when the nuclei in the synctium migrate out to the periphery and pole cells form at the posterior (germline cells)

Question 10

What occurs at the cellular blastoderm stage and what process follows this?

Answer 10

When cells form from the syncitial blastoderm stage

Followed by gastrulation

Question 11

How does the developmental process of insects differ significantly from other organisms?

Answer 11

Developmental pattern is set up in a synctium

• proteins can diffuse through the whole blastoderm
• molecules such as transcription factors can act as morphogens

Question 12

What is the importance of maternal gradients in the development of drosophila?

Answer 12

Maternal gradient - Bicoid mRNA

• when the drosophila egg is released from the ovary it already has bicoid mRNA tightly localised to anterior end, (and Nanos and Claudal at the posterior end)
• the Bicoid protein the forms a gradient along the AP axis (can do this because of the shared cytoplasm)
• regulates the expression of gap genes e.g. hunchback

Question 13

What is Bicoid?

Answer 13

• Bicoid is a homeobox transcription factor

- regulates the expression of gap genes e.g. hunchback

Question 14

What are the gap genes?

Answer 14

Hunchback, Giant, Krupple, Knirps

-code for transcription factors

Question 15

What experimental technique can be used to look for mRNA localisation?

Answer 15

In situ hybridisation

-label synthetic probe with complementary mRNA/DNA

Question 16

What experimental technique can be used to look for protein localisation?

Answer 16

Immunostaining using an antibody raised against different proteins
Or conjugate to a label (LacZ/GFP)

Question 17

Why is bicoid a morphogen?

Answer 17

Bicoid manipulates the hunchback gap gene

Question 18

How does bicod pattern hunchback expression?

Answer 18

• hunchback expression is an interpretation of the bicoid gradient
• expression is only activated above a certain threshold of bcd

Question 19

How can it be experimentally proven that bicoid patterns hunchback expression over a particular threshold?

Answer 19

Put in extra bicoid raising the concentration across the embryo

• as the protein diffuses further and stays above threshold much further along the AP axis
• results in an increase in the level of hunchback

Question 20

What is the activity of hunchback?

Answer 20

• transcription factor

- forms a gradient to which other gap genes respond

Question 21

How is krupple activated?

Answer 21

• gap gene
• activated by low levels of hb protein but repressed by high levels - expressed in a band in the centre of the embryo
• other gaps genes e.g. giant and knirps also repress krupple (much auto and cross regulation among gap genes)

Question 22

What are the main features of the gap genes?

Answer 22

• respond to maternal gradients and to each other
• mutations result in large continuous deletons of reions of the embryo
• expression spans large regions of the embryo
• edges of the regions of expression are graded and overalpping

Question 23

What do mutations in the gap genes result in?

Answer 23

-mutations result in large continuous deletons of reions of the embryo

Question 24

What are the pair rule genes?

Answer 24

-ftz and eve
code for transcription factors
-interpret the gap genes

Question 25

What do mutations in pair rule genes result in?

Answer 25

-affect homologous parts of alternate segments resulting in double segment periodicity defects

Question 26

What is the expression pattern of pair rule genes?

Answer 26

• initial expression is broad
• then stripes are formed by both accumulation of gene product in the stripes (continued and increasing expression) and a loss of gene expression in the intervening stripes (transcriptional repression and turnover)

Question 27

Where do the pair rule genes fall in the hierachy of genes involved in drosophila segmentation?

Answer 27

• intermediates between the non-periodic expression of gap genes
• and the repeated expression pattens of the segment polarity genes

Question 28

What is primary pair rule gene expression determined by? And what is secondary pair-rule gene expression regulated by?

Answer 28

• primary pair rule gene expression is determined by the gap genes alone
• secondary pair rule gene expression is regulated by other pair-rule genes

Question 29

What is the process by which stripes of pair-rule genes are formed?

Answer 29

Gap genes bind to one of the regulatory regions in the promoter of pair-rule genes e.g. even-skipped

• promoter has activation and repressor binding sites (these overlap)
• for even-skipped, Bicoid and hunchback are activators, Giant and Kruppel are repressors
• whether even skipped is expressed depends on whether more of the activators or repressors bind t that particular location in the embryo

Question 30

How can you experimentaly show the expression of gap genes due to the regulatory region?

Answer 30

• create a transgenic fly with a specific enhancer (e.g. a gap gene) driven lacZ
• when given substrate B galactoside, section that turn blue are controlled by that regulatory region

Question 31

What do high levels of ftz and eve control

Answer 31

the expression of segment polarity gene, engrail

Question 32

What are the features of the segment polarity genes?

Answer 32

-expressed when the embryo has become cellularised (no longer a synctium)
-not all segment polarity genes code for transcription factors (unline the gap and pair-rule genes)
-diverse group of gene, many coding for elements of signal transduction pathways
-

Question 33

What are teh features of the engrailed class of segment polarity genes?

Answer 33

• 14 stripes in a cellularised embryo
• expressed in each parasegment
• acts by fixing the positions of parasegment boundaires
• this establishes the ginal segment boundaries of the larval epidermis

Question 34

What results from segment polarity mutations?

Answer 34

• disrupt the normal A/P polarity

- phenotypes produced are mirror images or tandem duplications of segments

Question 35

What is the experimental evidence for the order in which Ftz and Engrailed act?

Answer 35

Fts and eve followed by engrailed
-used Ftz and eve mutants and molecular immunostaining to identify their localisations

SHOWED:

• Wt: 7 Ftz stipes and 14 engrailed striped
• Ftz mutant: lose Ftz stripes, only get alternate engrailed stripes
• En mutant: 7 stripes of Ftz, lose all engrail stripes
• Ftz is required for engrailed expression
• Ftz acts first

Question 36

What are the developmetnally important structures in the drosophila embro and how are these patterned?

Answer 36

Parasegments

• patterned by pair rule genes, gap gene and segment polarity genes
• offset slightly from segemtns

Question 37

How does wingless and engrailed pattern the segments?

Answer 37

• no ftz, no eve = wingless expression
• high ftz, high eve = engrailed expressed
• wingless expressed in the posterior most cell of the parasegment
• engrailed expressed in the anterior most cell of the parasegment
• anterior most cell of the parasegment develops in to the most posterior cell in the segments

Question 38

What is the interaction between wingless and engrailed?

Answer 38

Expressed in two cells that line up along the parasegment boundary

• most posterior cell of the first parasegment expressed wingless
• wingless protein is secreted from this cell and is the ligand for frizzled
• activation of the frizzled receptor begins a signalling cascade
• frizzled activated Dsh which surpresses the activity of Zw3
• this leads to higher levels of armadillo
• armadillo results in the transcription of engrailed, resultin in the activation of the expression of hedhog by the most anterior cell of the second parasegment
• hedgehod is secreted from this cell, and is the ligand for the patched receptors
• activation of patched results results in the transcription of wingless, through the smoothened protein and cubitus interruptus

Question 39

What is the purpose of the interaction between wingless and engrailed (and hedgehod)?

Answer 39

To maintain the parasegment boundary

Question 40

How do wingless and hedgehog result in morphogen gradients?

Answer 40

• increased levels of Hh converts more cells to more anterior cell types
• different cell fates depend on how much signal is recieved:
• increase in Hh signalling then 4* cells adopt a more anterior (2* or 3*) cell fate
• Hh initially acts to maintain Wg expression
• later it acts to pattern the parasegmnent in a concentraion dependent manner

Question 41

How does the feedback between the en expressing cell and the adjacent wg expressing cell maintain the compartment (parasegment) boundary?

Answer 41

• segment polarity genes wingless and hedgehog are signalling molecules
• wg maintains the expression of en in cells posterior to wg expressing cells
• en expressing cells activate the expression of Hh which signals to the adjacent cell and maintains the expression of wingless
• in the absence of wg, expression of hh and en is lose
• loss of segment polarity

Question 42

What does a loss of wg result in?

Answer 42

• loss of expression of both hh and en

- loss of segment polarity

Question 43

What is a morphogen?

Answer 43

A substance governing the pattern of tissue development in the process of morphogenesis
-produces specific cellular responsees depending on it local concentration

Question 44

Where are fts and eve expressed?

Answer 44

In the most anterior cell of alternate para segments

-form a gradient