W8

Question 1

What causes the imaginal discs to be formed?

Answer 1

Positional information from the D-V, A-P patterning genes.

Question 2

What do signaling pathways drive in leg imaginal disc development?

Answer 2

1. cell proliferation and 2. pattern formation in the leg imaginal discs during the larval stages. (distalless/Brista inner most of the concentric circles) 3. disc eversion: During pupation, the leg imaginal disc gets “telescoped out”, to generate the extended structures of the adult leg.

Question 3

What are the important features of the wing pattern?

Answer 3

1. The anterior wing margin is equipped with bristles (=microchaete: mechanosensory organs). 2. The first wing vein runs along the anterior wing margin.The other longitudinal wing veins (2nd, 3rd, 4th, 5th) are formed within the blade at defined positions.

Question 4

What are the posterior and anterior compartments of the imaginal discs?

Answer 4

The en-expressing subregion (en “on”) forms the „posterior compartment“. The other subregion (en “off”) the „anterior compartment“. These compartments are separated by a „border of clonal restriction“.

Question 5

How was the existence of compartments discovered?

Answer 5

clonal analysis- GFP/en-lacZ marking showed that cells did not cross the clonal boundary.

Question 6

Clonal analysis Method 1a

Answer 6

35S::Tag1-GUS transgene is used to randomly label cells during Arabidopsis development.

Question 7

Clonal analysis method 1b

Answer 7

removal of stop cassette from a transgene Act5C>stop>GAL4 > GFP Act5C - constitutive promotr GAL4 - yeast transcription factor As long as the stop cassette is present, there is no transcription. Stop cassette removed by heat shock.

Question 8

Clonal analysis method 2a

Answer 8

X-ray induced mitotic recombination

Question 9

Clonal analysis method 2b

Answer 9

Induction by site-specific recombination. For example by FLP recombinase. FLP recombinase can induce mitotic recombination when each of the two homologous chromosomes carry a centromere-proximal FRT site.

Question 10

Wing pattern formation

Answer 10

engrailed (en) encodes a transcription factor (Posterior). en expression results in hedgehog (hh) expression. The Hedgehog protein (Hh) is a secreted signaling protein which spreads out into the anterior compartment. Only the anterior cells along the compartment boundary are exposed to Hh levels that are sufficiently high to activate a signal transduction pathway (via Patched-Smoothened). As a consequence the expression of decapentaplegic (dpp) is activated in these cells. Another compartment boundary separates the dorsal (apterous expressing) from the ventral side (apterous non-expressing). Similar signalling processes (involving Notch signaling) result in the expression of wingless (wg) along this boundary.

Question 11

Dpp activates which genes in wing imaginal disk?

Answer 11

Omb at low levels, spalt at high levels.

Question 12

Dpp acts in a non cell-autonomous way. What is this?

Answer 12

Dpp acts on other cells than the one expressing Dpp.

Question 13

Which Hox genes determin the identity of the imaginal discs?

Answer 13

HOC-C for example: Antp but not Ubx expression in wing, Ubx but not Antp expression in the haltere.

Question 14

What are LSDS gradients

Answer 14

localized source and dispersed sink. The exact nature of these morphogen gradients are important for pattern details.

Question 15

clonal analysis – methods summary

Answer 15

Various genetic strategies (removal of stop cassettes in marker genes, mitotic recombination) can be applied for the induction of a heritable genetic change in single somatic cells. Usually they involve site-specific recombination (for example using induced expression of FLP recombinase recognizing FRT sequences; FRT = FLP recombinase target sequences in Drosophila, or spontaneous excision of the autonomous Tag 1 transposon in Arabidopsis). These genetic strategies produce genetic mosaics/chimaeras: not all cells of an individual are genetically identical. Clonal analyses can also be performed with dye marking instead of genetic marking (for example dye injection into a single cell at t = 0 and analysis at t = x). Past and present clonal analyses in various model organisms have made/are making major contributions to our understanding of developmental processes.

Question 16

summary: pattern formation in wing discs

Answer 16

1. Formation of imaginal discs:
   A-P and D-V patterning systems result in the specification of the imaginal disc region in the embryonic ectoderm. The identity of the invaginating imaginal disc is specified by the expression of HOX-C genes (for example: Antp but not Ubx
   expression in the wing disc; Ubx but not Antp expression in the haltere disc). Moreover, the invaginating disc inherits an anterior compartment (engrailed nonexpressing) and a posterior compartment (engrailed expressing) from the ectoderm.
2. Hh protein secreted from the posterior compartment induces Dpp expression in the anterior compartment along the A-P compartment boundary.
3. Dpp secreted along the A-P boundary spreads laterally and forms a morphogen gradient specifying pattern along the A-P axis by controlling the expression of target genes like spalt and omb over long ranges (> 30 cell diameters).
4. After imaginal disc formation, an additional compartment boundary is established, separating a dorsal (apterous expressing) from a ventral (apterous non-expressing) compartment. Notch signaling is involved in D-V boundary formation. Wingless is expressed and secreted by a stripe of cells along the compartment boundary.
5. Wingless spreads laterally and acts as a morphogen, patterning the wing margin region.
6. Dpp and Wingless are also involved in the regulation of cell proliferation.

Question 17

vertebrate limb development (2): overview

Answer 17

Limb development starts after gastrulation.
Limb bud formation:
Cell proliferation in the lateral plate mesoderm (see later) is not slowed down after gastrulation in the region where fore- and hind limb development starts, and a morphologically distinct ridge forms in the overlaying ectoderm (AER: apical ectoderm ridge). The resulting limb bud is therefore a bud of mesodermal cells underneath the AER.

Limb bud outgrowth:
Subsequently, the limb bud grows out, and pattern formation occurs during outgrowth. Proximal structures are formed before distal structures. Mesodermal cells condense regionally and form cartilage elements. These elements are subsequently converted into bones (from the center of the bone towards the end; epiphysis formation: growth zone at the end which allows postembryonic growth/length increase). Mesodermal cells derived from a part of the somites (myotome; see later) migrate into the limb bud and form muscles. The bone pattern directs the muscle pattern.

Question 18

limb bud formation: where?

Answer 18

Available evidence suggests that expression of particular combinations of HOM-C genes stabilizes Fgf10 expression in the prospective limb bud region (FGF: fibroblastderived growth factor). Signaling by Wnt family members (8c, 2b) is involved.

Forelimb buds form in the region of the anterior border of hoxc6 expression where the first thoracic vertebrae are formed. (Note that the number of cervical as well as other somites is different in mice and chick.)

Question 19

mutual stabilization of differential expression
of FGF family members in the limb bud

Answer 19

Fgf10 in the lateral plate mesoderm induces Fgf8 expression in the overlaying ectoderm which forms the AER (apical ectoderm ridge). Fgf10 and Fgf8 signaling between adjacent mesoderm (= PZ: progress zone) and ectoderm (AER) of the limb bud stabilize each other (positive feedback loops).

Question 20

conclusions from the experiments on limb bud formation

Answer 20

1. The AER is required for maintenance of the PZ (mesoderm underneath the AER) in a plastic proliferating state so that proximal to distal elements can be made in succession.
2. The AER is important for A-P patterning. (->The AER is required for maintenance of the ZPA: zone of polarizing activity)
3. The AER drives outgrowth of the mesenchym, but it does not specify the identity of the mesenchym. (-> Limb identity is specified independently by Pitx1/Tbx4 and Tbx5)
4. Not just the PZ is dependent on the AER (see 1.), but also reciprocally the AER is maintained by the PZ (Fgf10).
5. Fgf8 (and Fgf4) production is the crucial activity of the AER.

Question 21

linb bud proximal – distal patterning (P-D)

Answer 21

Removal of AER results in loss of proliferation in the progress zone, and premature termination of limb extension. Distal pattern elements are no longer formed. Accordingly, the “progress zone” model was postulated. However, not all of the genetic data is consistent with the progress zone model and AER removal also results in apoptosis within the PZ. The “early allocation-expansion” model was proposed as an alternative.

Question 22

limb bud anterior – posterior patterning (A-P)

Answer 22

Transplantation experiments revealed a zone of polarizing activity (ZPA, also called polarizing region) which acts as an organizer for patterning the anterior-posterior axis. The ZPA was later found to express and secrete Sonic Hedgehog (Shh), one of three human Hedgehog protein family members. The production of Shh is the crucial role of the ZPA. Beads releasing Shh mimick ZPA activity. Mutations in Shh affect A-P patterning. Shh appears to act as a long-range morphogen.

Question 23

summary: pattern formation in vertebrate limb
development

Answer 23

• Position of limbs along the AP axis of tetrapods is determined by the expression
  pattern of HOM-C genes, which direct expression of FGF10 in the lateral plate
  mesoderm where limb buds will form.
• FGF10 expression induces the overlaying ectoderm to generate the anterior
  ectodermal ridge (AER).
• Limb identity is determined by Pitx1 and the T-box transcription factors Tbx4 (hindlimb)
  versus Tbx5 (forelimb).
• Development along the P-D axis is controlled by the AER, which secretes FGF8 and
  maintains the cells in the underlying progress zone in an undifferentiated and proliferating state.
• Development along the A-P axis is controlled by the ZPA, which secretes the morphogen Sonic hedgehog (Shh).
• Pattern formation along the D-V axis involves Wnt-7a signaling from the dorsal side.
• Expression of HOM-C genes is involved in the specification of both P-D and A-P axes.
• Mutual interactions via signaling between the organizing centers AER (ectoderm), PZ (mesoderm), ZPA (posterior mesoderm) and the dorsal side are involved in their maintenance by positive feedback loops. AER: FGF8 and 4; PZ: FGF10; Dorsal ectoderm: Wnt-7a

Question 24

Zusammenfassung Visuelle Transduktionskaskade

Answer 24

-Licht aktiviert Photopigment (11cis Retinal wird zu all-trans Retinal)

• Rhodopsin aktiviert Transduzin (trimeres G-Protein)
• a Untereinheit des aktivierten Transduzin disoziiert und aktiviert cGMP Phosphodiesterase (PDE) Aktivierte PDE hydrolysiert cGMP
• cGMP gekoppelte Kanäle schliessen
• Zelle hyperpolarisiert und schüttet weniger Neurotransmitter aus

Question 25

Signaltransduktion in olfaktorischen Rezeptorneuronen

Answer 25