Steroid Hormonal Regulation of Development in Drosophila

Craig T. Woodard

Mount Holyoke College

20-hydroxyecdysone

Drosophila Life Cycle

How can a single steroid hormone How can a single steroid hormone elicit different responses at elicit different responses at

different times in development?different times in development?

Drosophila Life Cycle

Puffs

Early

2B5

74EF

75B

Prepupal early

93F

Mid prepupal

75CD

Genes

Early

BR-C

E74

E75

Prepupal early

E93

Mid prepupal

ßFTZ-F1

Edysone

BR-C

E74A

E75A

E93

ßFTZ-F1

Hours relative to puparium formation

Salivary Gland Developmental Northern Analysis

HypothesisA. ßFTZ-F1 provides the prepupal stage-specific

E93 early gene with the competence* to be induced by ecdysone

1) ßFTZ-F1 thus directs the stage-specificity of the E93 response to ecdysone.

B. ßFTZ-F1 provides the early genes, the BR-C, E74A and E75A with the competence* to be reinduced by the prepupal ecdysone pulse.

*Competence the ability to respond to an inductive signal

Hours relative to puparium formation

BR-C

E74A

E75A

E93

ßFTZ-F1

EXPERIMENTAL DESIGN

• Transformant Flies called P[F-F1] were used that express a high level of ßFTZ-F1 protein upon heat shock.

• Control w1118 and transformant w;P[F-F1] late-third instar larvae were heat shocked for 30 min. and then allowed to recover at 25˚ C for 2 hrs.

• Salivary glands were dissected.

• Total RNA was extracted from the salivary glands and analyzed for E93 mRNA by Northern blot hybridization. The Northern blot was also probed with rp49 (gene encoding ribosomal protein) as a control for loading and transfer.

w

w;P[F-F1]

Hours relative to puparium formation

BR-C

E74A

E75A

E93

ßFTZ-F1

EXPERIMENTAL DESIGN

• Transformant Flies called P[F-F1] were used that express a high level of ßFTZ-F1 protein upon heat shock.

• Control w1118 and transformant w;P[F-F1] mid-third instar larvae were heat shocked for 30 min. and the salivary glands were immediately dissected in oxygenated Robb’s saline.

• The salivary glands were then cultured in the presence of oxygen at 25˚ C for 2 hr with or without ecdysone.

• Total RNA was extracted from the salivary glands and analyzed for E93 mRNA by Northern blot hybridization. The Northern blot was also probed with rp49 (gene encoding ribosomal protein) as a control for loading and transfer.

ex17 is a Mutation in ßFTZ-F1

Expression of wild-type ßFTZ-F1 from a transgene

rescues ex17 mutants

Levels of early gene transcripts are reduced in ßFTZ-F1 mutant

prepupae

E93 transcription is greatly reduced in ßFTZ-F1 mutant salivary glands

control tissue mutant tissue

E93

rp49

E93

rp49

0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14

ßFTZ-F1 mutants fail to histolyze larval salivary glands

• Normal salivary gland histolysis

Results of ßFTZ-F1 mutations

• head eversion

• leg elongation

• wing extension

Mutations in ßFTZ-F1 disrupt leg morphogenesis

Cell Shape Changes During Leg Disc Elongation

Courtesy of Condic et al. 1991. Development 111:23-33

a b

Normal Leg Development

Comparative Leg Development

Control

ßFTZ-F1 Mutant

Possible Causes of Short Legs1) Contraction of the muscles is too weak in

ßFTZ-F1 mutants.

2) The pupal cuticle is too rigid by the time the muscles contract in ßFTZ-F1 mutants.

3) Connections to the puparium are not sufficiently weakened in ßFTZ-F1 mutants.

4) There is something wrong with the leg imaginal discs in ßFTZ-F1 mutants.

0102030405060708090

100

controluntreated

mutantuntreated

controltreated

mutanttreated

Leg Extension in ßFTZ-F1 Mutants can be Rescued by a Drop in Pressure

Percent of animals

with normal leg-length

(n = 27) (n = 20) (n = 11) (n = 22)

Possible Causes of Short Legs1) Contraction of the muscles is too weak in

ßFTZ-F1 mutants.

2) The pupal cuticle is too rigid by the time the muscles contract in ßFTZ-F1 mutants.

3) Connections to the puparium are not sufficiently weakened in ßFTZ-F1 mutants.

---------------------------------------------------------------4) There is something wrong with the leg imaginal

discs in ßFTZ-F1 mutants.RULED OUT

Possible Causes of Short Legs1) Contraction of the muscles is too weak in

ßFTZ-F1 mutants.

2) The pupal cuticle is too rigid by the time the muscles contract in ßFTZ-F1 mutants.

---------------------------------------------------------------3) Connections to the puparium are not sufficiently

weakened in ßFTZ-F1 mutants.RULED OUT

4) There is something wrong with the leg imaginal discs in ßFTZ-F1 mutants.

RULED OUT

Conclusions

ßFTZ-F1 mutants are unable to generate sufficient internal pressure (at the appropriate time) to extend their legs, evert their heads, and extend their wings.

We have been unable to detect ultrastructural abnormalities in the muscles thought to

generate this internal pressure.

Hypothesis - Perhaps there are defects in the neurons that innervate these muscles.

Testing the HypothesesHypothesis - There are defects in neurons that

innervate the muscles.

-Test by examining neurons, perhaps making use of animals expressing neuron-specific GFP.

Hypothesis - The pupal cuticle is too rigid by the time the muscles contract in the mutants.

-Test by aging the mutant and control animals a bit longer before exposing them to a drop in pressure

-Test by measuring the tensile strength of mutant and control pupal cuticle in staged animals.

Ecdysone, ßFTZ-F1, E93 and

Programmed Cell Death

(Tissue-Specificity)

ßFTZ-F1 is required for E93 transcription in larval salivary glands

control tissue mutant tissue

E93

rp49

E93

rp49

0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14

If E93 is required for a complete programmed cell death response, how

does destruction of the larval gut start at the beginning of metamorphosis (before

ßFTZ-F1 is expressed) ?

ßFTZ-F1 is not required for E93 transcription in larval gut tissue

mutant tissuecontrol tissue

E93

rp49

0 2 4 6 8 10 12 14

E93

rp49

0 2 4 6 8 10 12 14

IN WHICH TISSUES DOES THE EXPRESSION OF ßFTZ-F1 AFFECT

THE ECDYSONE INDUCTION OF BR-C, E74A, E75A AND E93

TRANSCRIPTION?

EXPERIMENTAL DESIGN

• Transformant Flies called P[F-F1] were used that express a high level of ßFTZ-F1 protein upon heat shock.

• Control w1118 and transformant w;P[F-F1] mid-third instar larvae were heat shocked for 30 min. and the various tissues were immediately dissected in oxygenated Robb’s saline.

• The tissues were then cultured in the presence of oxygen at 25˚ C for 2 hr with or without ecdysone.

• Total RNA was extracted from the tissues and analyzed for E93 mRNA by Northern blot hybridization. The Northern blot was also probed with rp49 (gene encoding ribosomal protein) as a control for loading and transfer.

RESULTS

•Northern hybridization results show that the induction of E93 by ßFTZ-F1 expression differs from tissue to tissue in mid-third instar larvae.

Induction of E93 by ßFTZ-F1in late-third instar larvae

CONDITIONCNS GUT IMAGINALDISCS

FAT SALIVARYGLANDS

w1118

[-Ecd] -- -- -- -- --w;P[F-F1][-Ecd] + -- -- + --W1118

[+Ecd] -- -- -- -- --w;P[F-F1][+Ecd] + + -- + +

FUTURE DIRECTIONSLegs, etc.- Attempt to rescue ßFTZ-F1-mutant defects by

ectopic expression of target genes.

Other Projects- Continue examining the regulation of target

genes by ßFTZ-F1 in specific tissues.

- Decipher the molecular mechanism by which ßFTZ-F1 provides target genes with the competence to respond to ecdysone.

Acknowledgements

• Mount Holyoke College• Tina M. Fortier**• Samara Brown**• Zareen Gauhar • Dana Cruz• Michael Chapman• Jennifer R. McCabe• Priya Vasa• Lynn L’Archeveque• Margaret Lobo• Emily McNutt• Tetyanya Obukhanych• Petra Scamborova• Diyya Mathur• Biology 340 Class!

• University of Utah

• Carl Thummel

• Eric Baehrecke

• Julie Broadus

• Bart Endrizzi

• Special Thanks for Technical Assistance

• Rachel Fink

• Diane Kelly

ßFTZ-F1 mutants fail to histolyze larval salivary glands

ßFTZ-F1 mutants exhibit pupal lethality and defects in

morphogenesis

Ecdysone concentrations

ßFTZ-F1

rp49

Ecdysone concentrations

Normalized RNA level

Edysone

BR-C

E74A

E75A

E93

ßFTZ-F1

Hours relative to puparium formation

Salivary Gland Developmental Northern Analysis