Lecture 1: AP patterning

Question 1

How is the bicoid gradient established?

Answer 1

Bicoid is one of the proteins used to pattern the A-P axis.
We can view this with mRNA probes.
• Or we can use Ab protein staining.
• Gradient is interpreted at least at 4 different levels (thresholds).
• Concentration dependent positional information. This is mediated by the bicoid protein. Bicoid is therefore a morphogen.
• Go from gradients to large blocks of expression.

Question 2

What are gap genes?

Answer 2

Gap genes are involved in the initial segmentation of the Drosophila. They are activated by maternal effect genes.
• The most obvious characteristic in resulting larvae is segments.
• We can screen for segmental phenotypes to find which genes lead to a developmental anomaly. 40,000 mutants created and there were 150 very clear segmentation phenotypes.
• One type of mutant found was gap mutants.
• In gap mutants, blocks of consecutive segments are deleted. Mutant phenotype is caused by a loss of gene activity.
• Gap genes are expressed in broad regions, they correspond to transcription factors.
• The mutants were linked to 4 main genes: giant, knirps, hunchback and kruppel.
• The mRNA for these genes can be shown as broad bands.
• Gap genes are activated at different concentrations of maternal effect proteins.

Question 3

What are pair rule genes?

Answer 3

Pair rule genes are expressed in alternating stripes which are used to define segments.
• Pair-rule genes are initially transcribed inaccurately.
• As transcription continues, the boundaries become more defined.
• The best examples of PR genes are even-skipped (eve) and fushi tarazu (ftz).
• Interactions between gene products sharpen expression boundaries.
• There are sharpening eve-ftz stripes in late development.
• There is autoactivation and mutual inhibition of the eve and ftz transcription factors.
• Pair rule stripes lead to segment polarity stripes.
• TFs can define areas surrounding the nuclei without cell membranes.
• Pair rule protein action define segments.

Question 4

What are segment polarity genes?

Answer 4

• Segment polarity genes are genes which are involved in segmentation.
• They induce the expression of downstream transcription factors in smaller blocks. In the end they cause the segmental expression of 14 segment polarity genes.
• Segment polarity genes are activated by pair-rule genes. They set up the anterior and posterior of each segment.
• Segment polarity mutants show identical defects which are repeated in every segment. This demonstrates that SPGs are required for pattern formation in each and every segment. These mutations can be deletions, duplications and axis inversion. A good example is the wingless mutant.
• SPG products are secreted signalling proteins as the embryo is now cellularized.
• The wnt gene encodes for the wingless signalling pathway.
• The engrailed gene encodes for the hedgehog signalling pathway.

Question 5

How do segment polarity genes function?

Answer 5

Wingless is only permitted to be expressed in one row of cells in each parasegment.
• Expression is defined by the expression of eve and ftz.
• Wingless acts on neighbouring cells to maintain the hedgehog expression.
• Wingless is activated in areas which have little or no Even-skipped or fushi tarazu protein.
• The en gene is activated by high concentrations of eve-skipped or Fushi tarazu protein.

Question 6

How is patterning maintained?

Answer 6

Once the patterning of parasegments is established, it must be continued as cell divisions occur.
• Wingless acts on neighbouring cells to maintain engrailed hedgehog expression.
• Hedgehog similarly acts on neighbouring cells to maintain wingless.
• Wingless also continues the maintenance of differentiated cells which express wingless.
• Wingless is secreted and it binds to the frizzled receptor which is expressed in cells expressing the en gene. The frizzled receptor activates the Wnt signal transduction pathway, which continues engrailed expression.
• The transcription pattern of these two cell types is stabilised.
• Protein diffusion is thought to prove gradients for the parasegment.

Question 7

How is the anterior-posterior gradient established?

Answer 7

Gradients of morphogens are used to establish the A-P axis.
• Bicoid corresponds to the anterior.
• Nanos corresponds to the posterior.
• Hunchback and caudal are distributed randomly at a high concentration.
• Bicoid has a 3’ UTR sequence which localises the mRNA by binding to dynein proteins. It is actively transported.
• Wherever bicoid is injected becomes the head.
• Nanos binds to the Oskar protein. The Oskar mRNA is transported to the posterior by kinesin. It acts as an anchor.
• Most nanos is not anchored. It is bound by translation inhibitors called smaug and CUP. Smaug binds to the 3’ UTR and recruits CUP which prevents translation. Oskar can dissociate CUP.
• Hunchback is repressed by Nanos.
• Caudal is repressed by bicoid.