Segmentation

Question 1

What is the main embryo D-V morphogen?

Answer 1

It is called dorsal. It determines the ventral face.

Question 2

What kind of protein is dorsal?

Answer 2

It is a transcription factor like the other morphogens.

Question 3

How is dorsal distributed in the embryo?

Answer 3

mRNA and protein are both evenly distributed throughout the embryo.

Question 4

How is a gradient of dorsal established?

Answer 4

The amount of active dorsal determines which side is active. It is sequestered in the cytoplasm of the dorsal side by a protein called cactus.

Question 5

What does the dorsal gene do?

Answer 5

On ventral side of the embryo it enters the nucleus and activates production of dorsal protein. The gradient of dorsal TF creates a gradient of spatzle and Toll gene products on the ventral side.

Question 6

What do spatze and Toll do on the ventral side of the embryo?

Answer 6

They help determine the dorso-ventral axis, which is determined by transcription factor Dorsal, which in turn is determined by the gradient of spazzing and Toll proteins deposited during egg formation.

They trigger a signal transduction pathway in cells that releases dorsal, migrates to nucleus, and activates genes for ventral fates.

Question 7

How is dorsal activated?

Answer 7

dorsal is inactivated by cactus.

TOLL on embryo surface binds to SPZ ligand which phosphorylates cactus and releases dorsal allowing it to move to the nucleus.

Dorsal activates transcription of zygotic ventral patterning genes.

On dorsal side the dorsal stays attached to cactus, therefore in cytoplasm

Question 8

What is the pathway of dorsal release from cactus?

Answer 8

SPZ ligand binds to TOLL. TOLL activates PLL which phosphorylates CACT-DL releasing DL and this results in DL exerting its effect in the nucleus. CACT = cactus DL = dorsal

Question 9

What happens if TOLL is mutated?

Answer 9

There is no way to release dorsal from cactus and so there will be no entry of any dorsal into any nucleus.

Question 10

What happens if cactus is mutated?

Answer 10

There is an inability for dorsal to be held away from the nucleus by cactus and so that results in all cells having dorsal expression both ventrally and dorsally.

Question 11

What does dorsal do in the nucleus?

Answer 11

It leads to mesoderm production most ventrally and then ectoderm deeper to it. I.e it leads to localisation of activity.

Question 12

What are the first zygotic A-P genes released?

Answer 12

gap genes. Kruppel and knirps (mutants have gaps in normal segmentation) promoters have differential sensitivity to the maternally derived TFs like bicoid, HB-M, and Caudal.

Question 13

What are the targets of gap genes?

Answer 13

There is a bifurcation of function: One branch establishes correct number of segments Another branch assigns proper identity to each segment.

Question 14

What do kruppel and knirps genes do?

Answer 14

They are important for large numbers of segments to be produced.

Question 15

How are the pair-rule genes activated?

Answer 15

By the gap genes and they are responsible for the production of stripes of segmentation. (on-off, on-off, etc)

Question 16

What do segment polarity genes do?

Answer 16

They create defined borders between segments by defining anterior and posterior aspects of each segment.

Question 17

What is the hierarchy of segmentation?

Answer 17

1. Maternal effect genes, then zygotic
2. Gap genes: divide embryo into broad regions
3. Pair-rule genes: divide embryo into stripes, defining segment borders
4. Segmentation polarity genes: divide segments into anterior & posterior halves
5. Homeotic selector genes: specify the identity of each segment.

Question 18

How are gap genes activated?

Answer 18

They are the first zygotic genes to be expressed along A-P axis. Mother sets up gradient of bicoid mRNA. Bicoid activates HB-zygotic which activates giant, kruppel, and knirps.

The A-P gradient of Bcd and Caudal regulate Kinross, Hunchback, Kruppel and Giant

Question 19

What does kruppel respond mostly to?

Answer 19

High levels of HunchBack in a concentration-dependent manner. High levels of HB in anterior suppress kruppel. Intermediate levels of HB activate kruppel. Low levels of HB fail to activate kruppel. So kruppel is most active in the middle. hence the segmentation they produce.

Question 20

What do pair-rule genes do?

Answer 20

Expressed in alternating bands resulting in zebra like pattern that divides the embryo in 14 stripes as a result of gap gene expression. The genes are activated in every second parasegment in the thorax and abdomen.

(Note the stage of the zygote is syncytial blastodermerm - still not cellular, but occurs after this)

Question 21

What pair-rule genes are expressed?

Answer 21

3 primary pair rule genes: hairy, even-skipped, and runt. 5 secondary genes. (including fushi tarazu)

(Even-skipped defines odd-numbered parasegments , Fushi tarazu expressed in even-numbered parasegments, Expression is only a few cells thick)

Question 22

What do Hedgehog genes do?

Answer 22

Allow for differentiation between left and right sides of the body.

Question 23

How are homeotic selector genes regulated?

Answer 23

The pair-rule and gap genes interact to regulate the homeotic selector genes.

Question 24

What do homeotic genes do?

Answer 24

Encode TFs and control segment identity

Question 25

How do homeotic selector genes regulate other genes?

Answer 25

All the homeotic selector genes contain the homeobox domain. Homeodomain allows TF to bind DNA. Proteins containing homeodomains may be activators or repressors of transcription. Not all homeobox-containing genes are homeotic genes that are associated with development.

Question 26

How many hom/hox gene clusters are there in flies and humans?

Answer 26

Flies: 1 cluster in insects Humans: 4 Hox clusters in mammals. • Homologs of drosophila selector genes have been discovered in virtually all other animals • Note: All expressed in the same direction, anterior expression is at 3’ end of the cluster.

Question 27

What are the three important maternally-derived proteins which provide signals neccessary for dorso venteral patterning?

Answer 27

- Toll - Spatzle - Dorsal

Question 28

Where is activated dorsal protein found?

Answer 28

Activated dorsal found in a gradient from dorsal region (LOW) to ventral (HIGH) surface Dorsal is in the nucleus (& active) ventrally Dorsal is in the cytoplasm (& inactive) in dorsal regions Active dorsal protein, expressed highly in cells of ventral midline.

Question 29

What are the similarities and differences between the basic body plan for a fly and vetebrates?

Answer 29

Similarities: -Anterior head Nerve cord running from anterior to posterior -Gut also running anterior to posterior Differences: Arthropod nerve cord is located ventrally Vertebrate cord is located dorsally the D-V axis is inverted

Question 30

Subdivision of dorso-ventral axis into regions by nuclear dorsal protein activates z——— g—— e———

Answer 30

Zygotic gene expression

Question 31

How do the cells interpret the positional information (i.e maternal A-P and D-V gradients) in the oocyte?

Question 32

Why are the gap genes called gap genes?

Answer 32

Because mutations resulted in large « gaps » in the body plan

Question 33

Understanding stripe formation: how does eve stripe 2 form?

Answer 33

• Binding of Bicoid and Hunchback proteins stimulates transcription of eve. • Binding of Giant and Krüppel represses eve transcription. • Trapped in a valley between high levels of the giant and Krüppel proteins, expression of eve in the second stripe is possible

Question 34

What are segment polarity genes?

Answer 34

* The combined expression of the pair-rule genes regulate transcription of the segment polarity genes -(e.g Engrailed expression is the result of the combined expression of pair- rule genes – including fushi tarazu and eve) * Are expressed in patterns of 14 stripes . Define the boundaries and polarities of the 14 segments * In contrast to Gap and pair-rule genes, they are not all transcription factors, have more diverse functions * Expression begins after cellularization

Question 35

Give two examples of segment polarity genes.

Answer 35

• engrailed (en) & wingless (wg) • Both genes encode constituents of the Hedgehog & Wnt signalling pathways • en& wgmutations both affect wing development in adult, but are expressed in many times/places. Within each segment - polarity is required for formation of segment boundaries; a series of genes is repeated in each segment. en (engrailed) and wg (wingless) are factors at the anterior and posterior of each segment.

Question 36

How were homeotic selector genes discovered?

Answer 36

Were discovered following observations of unusual mutations in drosophila where entire segments were converted into others • deletion of ultrabithorax turns 3rd thoracic segment into a 2nd segment - Complete identity change – fly has four wings, not two • Misexpression of antp in anterior segments results in legs forming where the antennae should be • “Homeosis” is the development of one body part with the phenotype of another e.g. antennapaedia mutant.

Question 37

How do we know developmental strategies in animals are ancient & highly conserved. What are the two examples?

Answer 37

Many Hox genes have now been knocked out in mice, similar phenotype to null flies 1. "Knockout" of HoxC8: enlarged vertebre HoxC8 KO vertebrae has transformed segmental identity to more anterior. Same as Ubx mutant in flies (i.e extra rib is related to extra wings) 2. HoxA13 and HoxD11-13 important for correct digit formation