Steroid Hormonal Control of Development in Drosophila Craig T. Woodard Mount Holyoke College

20-hydroxyecdysone

Drosophila Life Cycle

How can a single steroid hormone elicit different responses at 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

Hypothesis A. 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 w 1118 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 w 1118 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 tissuemutant tissue E93 rp49 E93 rp

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 Development 111:23-33 ab

Normal Leg Development

Comparative Leg Development Control ßFTZ-F1 Mutant

Possible Causes of Short Legs 1) 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.

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 Legs 1) 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 ) There is something wrong with the leg imaginal discs in ßFTZ-F1 mutants. RULED OUT

Possible Causes of Short Legs 1) 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 ) 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 Hypotheses Hypothesis - 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 tissuemutant tissue E93 rp49 E93 rp

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 rp E93 rp

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 w 1118 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

FUTURE DIRECTIONS Legs, 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