Development lecture 4

Organiser grafts showing inductin

Experiement: graft tissue from the dorsal side ontot hte ventral side

Result: Induction of a secondary axis

Where does the information come from?

1. From outside→ environmental factors (gravity, sperm entry point)

2. From inside→ maternal determinants put into egg cell

3. From outside→ cells signal to each other (induction)

   • after having been made different by maternal determinants

kidney

But how to find the machinery needed for induction

• Need to find the different signalling pathways and regulatorys of gene expression

→ Genetic screen→ to find zygotic genome stuff

some definitions for the experiemnt

• DTS→ dominant temperature sensitive

  • 29 degrees→ kills flies or makes them infertile

  • easy way to get id of DTS carrying progeny

• b→ balancer chromosome

  • lethal when homozygous and with clear markers

    • e.g beta-gal or GFP expression

    • or → curly wings for adults

How do this?

1. Treat a male with mutagen (EMS) (a/a)→ to form a* mutation sperm

2. Cross with a DTS/b female→ viable at 29 degrees

3. approx 5000 individual males outcrossed→ no. required for saturation of chromosome with mutations

   • i.e the mutant sperm from og fly→ mutant flies cross with more DTS/b female

4. Grow this F1 progeny→

   • b/b is embronicaly lethal

   • DTS/b and a*/DTS dead at 29 degrees

   • a*/b and a*/b mmale nad female surive!

5. Cross these a*/b x a*/b

   Result:

   • a*/a*→ embryonic lethal (with pattern defect?? that can be studied)

   • a*/b→ heterozygous viable shock

   • b/b→ embryonic lethal

What does the a*/a* show?

• Denticle patterns that develop on the ventral of each segment of larvae

• Used to look for aberrations in the normal pattern of development

→ THEREFORE→ find genes for each place in the larvae??

What ere the three patterns they found and their genes?

What are denticles used for?

• feed

• for larva to crawl on substrate in peristalsis

Therefore what did the mutants screens show?

• mutations that disrupt this pattern

• discovered gene beeded for normal development

and THEREFORE

→ Could show the gene encoded for:

• elements of signalling pathways

  and

• regulatory networks controlling gene expression

Example of a mutant screen

• found a gene for wingless signalling pathway

→ found many factors required to build larvae

Why this was important in overal research of development

• toolkit is largely conserved

• same genes used to built a fly could build a fish

→ therfore→ studing fly found many basic mechanisms to contruct humans

• why model organisms are really useful!

How did these mutatns interact with eachother

Formed a hierachy

→ showed that from intiail egg polarity→ gradual incrase in complexity to divide up the embryp into a large number of repeating units

How is the gene expression regulated

Expression of eve stripe 2:
Respressor→ giant and Kruppel

Promotor→ bicoid and hunchback

Result→ get a peak of eve when there is low giant and kruppel but also high bicoid and hunchback

Overall: different cobinations of promotors and repressors→ to narrow down the effect of a specific region

Example of conserved genes in Drosophila to vertebrates

Drosophila→ hegdeghog

• mutant→ embryo has a prickly pattern on dentricles

• encodes a diffusable signalling molcule needed for patterning the segment

Vertebrate→ sonic hedgehog

• homolgous gene

• key signalling molecules for vertebrate embryos

  • e.g central nervous system

  • limbs

Sonic Hedgehog in developing limb of a chick embryo (or mouse/human)

• In situ hybridisation dark staning→ shows SONIC HEDGHOG transitps

• expressed by cells at the posterior margin of the limb bud

• so Sonic hedgehog diffused anterioroly as a graded signal

→ This is critical for patterning of the limb in this axis!

So we know have information in the cytoplasm that regualted zygotic genes to lay out he ground plan for the embryo but now…

…We need additional mechanism which assign cells to particular pathways of differentiation bthe position that they have been assigned to!

The question to answer is

How do cells interpret positional information in the developing embryo?

French flag Model

1. Organisms develop into groupd (fields), separated by boundaries (which is controlled by what is above)

2. Each field will be dedicated to making a part of the final strucutre e.g arm or leg→ they have gene unlcoked for specific parts

3. Fields generate information about the position→ A gradient of a diffusible substance established between the boundaries

4. Cells the assess their position by reference to the local level of the gradient→ differentiate accordingly

5. All fields generate the same information BUT cells of each field have access to a different part of the genetic programme!→ so the same gradient does not mean the same limb will be generated

Even though the positional information across the field is the same

• Different fields have access to different parts of the genome

This is why→ in the diagram→ yess the positional information is the same→ so forms a flag of three colours in the same positions

BUT

• Because the fields are different in each, the have access to different parts of the genome

• → OVERALL: make different colours→ different flags!

Overall two aspects of patterns which are working

1. Original field genetic assignments

2. Positional information

Example of evidence for this

• The limb development of the chick

Why this is good evidence

→ the development of chick and mouse limbs are similar-. very ancient pathways!!

Axis of the chick limb

1. Proximal to Distal (closest to body→ away from the body)

2. Anterior→ Anterior

3. Dorsal→ Ventral

Testing French flag Model→ Prediction

Two fields of cells making different flags will have the same positional information

THEREFORE we predict

• Cells from one group transplanted to the other

• Will read new position

• Differentiate appropriately

BUT

• STILL following the programme to which they have previsouly been assigned

E.g explanation of this

1. Part of bottom unition flag put in top of french flag

2. Read its new position

3. STILL develops as a union flag

4. BUT→ as the top of the flag, instead of the bottom

Actual experimental evidence of this in chicks!

Experiment

1. Proximal leg

2. put into

3. Distal wing

Result:

• Forms distal leg

→ Matches new position but develop as ind limb

Conclusion:

1. cells are assigned in groups to make different parts of the organism

2. psotional information in each group in the same→ must be universal cues but genetic code they have access to is different

Next question to ask

Hows is this positional information generated?

How are elements laid down in a proximal to distal sequence: the zones

• Progress Zone→ region of dividing cells at tip of limb bud

• Apical ectodermal ridge (AER)→ most distal layer of ectodermal cells

  • The most distal region!

How do cells position in the porximo-disstal axis

• Measuring the time they spend in a region of dividing cells at the tip of the limbs

  → progress zone

Evidence for this?

• Removes the Apical ectodermal region at different time intervals

Results:

• development of the limb stopped at different stages of development

Conclusion:
- once leaves the progress zone, stop dividing and differentiate, laying down elements of the pattern in a proximo-distal sequence

What happened if transplanted an AER from a mouse?

• Recovered the limbs

→ shows conserved mechanism!

What in the Apical Ectodermal Region maintains proliferation?

Fibroblast Growth Factors (FGFs)

→ The more distal the part of the limb→ the most time it has spent in the AER

How we know the FGFs do this?

• Even if AER is removed

• the FGFs are sufficent to elicit cell division

How is Antero-posterior axis patterned?

• Layed down by measuring their position to the zone of polarising activity ZPA

  • This is the posterior region

What does the ZPA do to cause these patterned?

1. Generates a gradient of a substance

2. Diffuses across the limb

3. cells respond according to their level in the gradient

Experiemnts to show this

Transplanting the Zone of Polarising Activity ZPA

1. Add extra ZPA to the anterior margin

   → Cell differentiate to make mirror image duplication of the posterior digits

2. Add small amount to the anterior margin

   → Get a duplication of the small anterior limb but not symmetric→ does not reach high enough levesl to form another posterior type part of the limb

What we now know is the gradient signal

→ Sonic Hedgog synthesised by the cells in this region

However we now know the two signalling centres that organise limb bud development and what variable they are based on

Poximo-distal

• FGFs from the AER

• time based

Antero-posterior

• Shh from ZPA

• spatial based

Parasite in frog→ example of limbs buds behaving like self-organised organs

• Example of how one mutation/paraite

• Can cause rise of self-contained organised limb

WHY?→ limb buds have features of self-organising organs