Development Lecture 3

How do we find out what is present in egg cells

Must all be from what mother put in:

• Systematically create mutations in the maternal genome

• screen for mutations that lead to the production of defective eggs and embryos

  → identify all the genes concerned

NEXT: show how their products operate in egg cells

Experiment hypothesis and test

HYpothesis: the egg contains fine-grained map of future cell fates in the form of cytoplasmic determinants

Experiment: test the genome of the mother→ she is responsible for making the egg cell→ use mutations

What are these mutants called

Maternal Effect Mutations

• mutations that have their effect only when they are present in the mother

• NO effect if they are carrier by the father

Female mutation x male wild type=

Mutation/wild type defected embryo

Female wild type x male mutation=

mutation/wild type normal embryo

Results

• Not a fine-grained map

Examples of mutant phenotypes recovered in a screen for maternal mutations in Drosophila

• Bicoid→ anterior strucutures

• nanos→ posterior strucutures

• torso→ terminal

• In each case→ embryos/larvae produced LACK these particular strucutures

What does the bicoid encode for

• maternal determinant for anterior strucutures

How can we test how genes like bicoid act

• Cytoplasm transfer

How this works

1. transplnatation of anterior cytoplasm

→ rescues and induces anterior strucutures in bicoid mutants

How we know that bioid encodes something for the anterior?

1. if anterior transferred to the middle of the bicoid mutant egg

2. the head forms in the middle!

What do the gradients in the diagram suggest?

SHow the active substance→ bicoid protein

• localised in the anterior part of the egg cell

• this shows that not only is bicoid protein necessary but also sufficient in forming anterior/head in the cell

How was it confirmed that it is localised to the anterior part of the egg cell?

Visualisation (in situ ybrisiation)

• of bicoid RNA and protein in the egg cell

→ RESULT: localised to the anterior pole and diffusing away from it in a graded fashion

• Shows that bicoid works as a switch. Once there is enough of it to get to a threshold→ switches on the hunchback gene to half of the cell

Overall how does bicoid work?

Bicoid codes for transciption factor

1. maternalling generated bicoid mRNA deposited at anterior tip

2. translation leads to gradient of Bicoid protein

3. Bicoid protein leads to activation anteriorly of zygotic gene→ hunchback (i.e working as a trasnciption factor)

Bicoid is ina gradient

over a line→ causes a straight line of hunchback

If bicoid is sufficient to trigger anterior formation, how many other such factors does the maternal genome encode and put into the egg?

• Does the egg contain a fine map of determinants for all the future strucutures of the larva?

Do a maternal screen for mutations:
RESULT: not such map, instead:→ 4 classes of mutants

What are the 4 classes of determinants

1. Formation of germ cells→ oskar

2. anterior-posterior axis→ biocoid

3. Dorso-ventral axis→ dorsal

4. termini of the embryo

Overall, what does this say about the role of the maternal genome?

• sets coordinate for future development

• laying out the a-p and d-v axese

When are the finer details (assignment of cells to form particular strucutures), made?

After the Mid-Blastula Transition

• as the zygotic genome becomes active!

How does maternal productions form dorsal-ventral axis in Drosophila

1. Trasnciption facotr Dorsal

2. leads to activation of the zygotic gene twist

3. in the nuclei of the most ventral cells

4. Expression of twist→ ventral cells to form medoerm

But where do these transcription factors know where to go?

• need to look back in the maternal material

• and how the egg is made

How are bicoid and askar used at different ends? (how are they localised)

1. the mRNA of both segregate to oppsotide ends

2. via microtubules of the oocyte

3. bicoid mRNA→ anterior

4. oskar→ mRNA to the posterior

What does the specificity of this interaction depend on?

• 3’ untranslated regions (3’ UTR) of the transpits

How do we know this?

Experiment:

1. oskar transgene fitted with bicoid 3’ UTR sequences

Result:

• oaskar mRNA localised with bicoid mRNA

• pole cells then form at the anterior end

How do you polarise an egg?

• how do you start to get the singularities in an otherwise uniform cell

• from which subsequence differeneces then unfold

Example 1→ Fucus

Example 2→ Xenopus

E.g 1: Fucus→ facts

• Common sea weed Fucus

• Sheds eggs into sea water→ which are fertilised

E.g 1 Fucus→ what happens to the egg?

1. uniform sphere for the first few hours

2. polarises:

Rresult after first cell division:

• one Apical cell→ produce the frond on the sea weed

• one Basal cell→ make the hold fast

E.g 1→ how is this apico-basal polarity set?

• cell responds to environmental cues

  • Light, pH. gavity

• redistributing pumps and leaks of Ca2+

OVERALL→ uniform flux of Ca2+ across the membrane→ beomes focussed as the current flowing in the apino-basal axis

Small initial differences in membrane charge can be amplified by positive feedback loops

E.g 1: Fucus polarity

1. light (or other external signal)

2. causes electrical depolarisation→ causes the apical to become negative and the basal to become positive

3. Accumulation of charge Ca2+ pumps and leaks

4. Ca2+ enters the basal and leaks out of the apical

5. Overall reinforces itself→ amplification

6. Waves cause recruitment of material at the basal pole

7. Causes asymmetric cell division

OVERALL→ differentiated directly,

12 hours→ fast!

Example 2: Xenopus→ the two axes

1. Animal-vegetal axis

2. Ventral-dorsal

→ Seen on the egg as it has camoflage a bit like the frog itself→ dark on top and light on the bottom

Axes before and after gastrulation

Before gastrulation:

• dorsla-and ventral look like the anterior and posterior but they are what will become the dorsal and ventral!

1. Animal-vegetal A-V axis

Animal hemisphere

• hald of an egg/ embryo that contains less yolk

• divides more rapidly compared to vegetal

In eggs with considerable yolk

• animal hemisphere will be the upper half

Why?→ what is the environmental cue?

Gravity

1. Egg floats in the water

2. heavy yolk falls to the lower pole

3. les yolky cytoplasm towards the animal pole

What actually sets the animal-vegetal axis

mRNAs localising due to gravity

How is this shown in experiments?

Centrifuging or shaking eggs

1. transcipts like VegT RNA→ associated in the region with the yolk at the vegetal pole

What did this experiemnt also show?

• Wnt11 RNA→

  • closer to the plasma membrnae in the cortical cytoplasm

  • more freely able to move

→ This hints to its use in polarising role for the doral central axis!

2. How is the dorsal ventral axis polarity made?

1. Environmental stimulus→ Entry of the sperm

2. triggers mechanism→ rotates the cortical cytoplasm 30 degrees away from sperm entry

3. vegetally located transcipts like Wnt11 are shifted away from entry point

4. Asymmetric redistribution→ sets future dorso-ventral axis

   • How?→ through the overlaying of the different signals

• dorsalising signals→ cortex is just inside the cytoplasm

• but the vegt does not move

• roates to be opposite side from where sperm enter

Which parts rotate→ how is this shown?

• The cortex against the underlying cytoplasm

BUT

• The stuff above does not (the yolky core)

Shown by→ microtubules

• Many mitotbules at the bottom for rotation→ not so mant at the apex→ which stays the same

• The diagram shows how the bottom Wnt11 rotates

• BUT

• the VegT do not roate ontop of this inthe yolky core

Microtubules

What are the finer details of thegene expression dictated by?

• by the zygote

• once the embryp has cellularised

What dictates the patterns of cell differentiation at this stage?

See image→ how do the cells in the marginal zone (which makes blood, somites heart etc)→ know that they have to make mesodermal tissues??

• cell-cell interactions→ shown in the fate map of amphibians

Experiment→ took apart each part of the egg cell

Conclusions

• Cells that are found are in contact with animal and vegetal cells

• Form into the mesoderm cells

→ The ones on their own→ just ectoderm or endoderm

Suggested that there are no mesodermal determinants but that they are just incontact?

Mesoderm only when two halves of the embryo come into contact→ INDUCTION→ EXTRINSIC!

Next experiment to test this

Experiment

• Put animal cap cells directly onto vegetal cells

• transplant

Result

• cells from the most animal pole can be

• Induced mesodermal tissue!

Conclusion:

• vegetal cells are the source of signals that ‘tells’ animal cells to make mesoderm

What are the signals involved?

• e.g Vg1 (used earlier to transcitp the vegetal half)

• encodes a signal of the TGF beta family of growth factors

What is the blastocoel

• fluid filled cavity in the embryo

Gastrulation

• Tissue movements which turn 2D→ 3D

1. Bottle cells move inward to form the doral lip of the blastopore

2. archenteron and cell migrate into the embryo

3. Moves inside out!

4. Displaces the blastocoel

5. Well established cellular movements in frog gastrulation:

   • involution

   • conergent extension

     • converge back to back which enxtends them

   • epiboly

6. AT THE END:

   • blastocoel gone

   • embryo surrounded by ectoderm

   • endoderm is internalised

   • mesoderm is between these

Overall result of gastrulation

• shifts the germ layers into 3D shape:

  • ectoderm on outside

  • then mesoderm

  • then endoderm interntalised

• No more blastocoel

• Archenteron made→ forms the gut