lec 9 - drosophila limb development

The core question: how does a tiny flat disc of ~30 cells grow into a perfectly patterned 3D wing (50,000 cells, 4 days later)?

morphogen gradients along two perpendicular axes

what is an imaginal disc

• small sacs of epithelial cells

  • float in larval body through metamorphosis (when most larval tissue is destroyed), and then unfold into adult structures

how do wing discs unfold into adult structures

wing disc eversion

what are the parts of the wing disc

• noctum

• dorsal proximal wing

• wing pouch

• ventral proximal wing

• pleura

describe the process of wing disc eversion

how are imaginal discs patterned

orthogonal morphogen gradients (AP/DV)

• cells read orthogonal morphogen gradients like coordinate planes

the AP gradient is patterned by

Hh, Engrailed (Eg), and Dpp

what happens when Shh is placed ectopically in the limb bud

chick: mirror image wing

• fly: extra wing

  • human: polydactyly

the DV gradient is patterned by

Wg/Wnt, Apterous (dorsal TF)

____ defines the posterior segments

Eg

how does Eg defining the posterior segment tranlate into the imaginal discs

when cells invaginate into imaginal discs, they inherit the posterior expression of Eg

how do we know Eg patterns posteriorly

reporter genes

• replace Eg gene with LacZ genes → LacZ is under the control of Eg enhancers

• when washed with soluble colorless X-gal, LacZ converts it to blue precipitate

• posterior wing is blue - therefore Eg is posterior

What is Dpp

• BMP protein

• fly TGF-B homolog

• Hh transcriptional target

• morphogen

what is the process of AP axis patterning

• posterior Eg activates Hh production but restricts Ci

• Hh diffuses over the compartment (AP) boundary into the anterior section - where there is Ci

• Hh pathway is activated → Patched receptor production is activated → Patched expresses at the AP/compartment boundary

• Hh activates Dpp, a morphogen - spreads outwardly from the compartment boundary

Dpp pathway

TKV/Pnt

Type 2 and Type 1 Dpp receptor heterodimer

Mad

Dpp TF when phosphorylated

Omb, Sal

Dpp target genes (Dpp activates)

brinker

Dpp target gene (Dpp inhibits)

How do we know Dpp is a morphogen?

1. antibodies for p-Mad show Dpp diffusion

2. when Dpp is tagged with GFP, can visualize the diffusion - area of protein is much wider than the region Dpp is produced in

3. high [Dpp] → Omb and Sal activation, but low [Dpp] → only Omb activation

   1. french flag model - changes in [Dpp] → changes in gene expression = morphogen

4. when GDP-Dpp is bound to the AP axis cell membrane by anti-GFP nanobodies, brinker (only active when Dpp is not present) expression is able to move closer to the AP axis than it normally does— implies that Dpp diffused outwardly and induced a certain cell fate

Clone 1: Eg^- cells cloned and transplanted into posterior region

Eg^- → increased Ci but decreased Hh → Hh from surrounding cells diffuse into cloned cell area → Dpp signaling only in cloned area

Clone 2: Eg⁺ cells are cloned and transplanted in anterior region

Eg⁺ → increased Hh but decreased Ci → Hh diffuses outwardly → increased Dpp signaling around the cloned cell region

Clone 3: Patched^- cells are cloned and transplanted in anterior region

Patched^- → Smo^- → increased Hh signaling → increased Dpp signaling in and around cloned cell area

Clone 2/3 (Hh/Dpp signaling in anterior wing disc) - what is the phenotype and why

phenotype: mirror image wing

why: because the Hh signaling pathway was activated so far from the compartment boundary, nearby cells believed that they were close to the compartment boundary and thus began patterning as such.

how is the DV axis patterned

• Apterous expressed dorsally - Wg signaling restricted

• Wg expresses at DV boundary

How do we know that Wg is diffusion/a morphogen

expression domain much tighter than protein location