Steroid Control of Leg Development in Drosophila

Craig T. Woodard

Mount Holyoke College

Drosophila Life Cycle

•The fruit fly undergoes complete metamorphosis.

•Development lasts 10-12 days during which the fly embryo develops into larvae, pupa and ecloses into an adult.

• Controlled by steroid hormone ecdysone

20-hydroxyecdysone

EcdysoneUltraspiracle

(USP)EcdysoneReceptor (EcR)

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

Ecdysone directs metamorphosisPuparium formation

Prepupal- pupal transition

•High titer of ecdysone at the end of 3rd instar larva initiates entry into metamorphosis

• Second high titer at approximately 11 hours APF initiates the Prepupal-Pupal Transition, which includes formation of adult body parts by morphogenesis and destruction of larva body parts through apoptosis

Morphogenesisof Adult Body Parts

Destruction of Larval body Parts by Programmed Cell Death

Gas bubble translation

Beginning of imaginal disc

morphogenesis

Stages in Drosophila Leg Development

Embryonic StageLeg imaginal discs patterned

Puparium Formation (Beginning of Metamorphosis = 0-Hrs. APF)Ecdysone induces Leg imaginal Discs Eversion and

Elongation

Prepupal-Pupal Transition (~12-Hrs. APF)Ecdysone induces Pupal Ecdysis, inflating and Extending Legs

Ecdysone directs leg morphogenesis during metamorphosis

Puparium formation (0-Hrs. APF) Pupal ecdysis

(part of the Prepupal- pupal transition)

•High titer of ecdysone at the end of 3rd instar larva initiates leg imaginal disc Elongation and Eversion

• Second high titer at approximately 10-12 hours APF initiates pupal ecdysis, which drives leg Extension, and other morphogenetic events of the Prepupal-Pupal Transition

Leg ExtensionLeg disc

Elongation and Eversion

Third Instar Larva

Leg Disc Elongation and Eversion

Adult

Ecdysone directs leg morphogenesis during metamorphosis

Puparium formation (0-Hrs. APF) Pupal ecdysis

(part of the Prepupal- pupal transition)

•High titer of ecdysone at the end of 3rd instar larva initiates leg imaginal disc Elongation and Eversion

• Second high titer at approximately 10-12 hours APF initiates pupal ecdysis, which drives leg Extension, and other morphogenetic events of the Prepupal-Pupal Transition

Leg ExtensionLeg disc

Elongation and Eversion

Normal Leg Development

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Third Instar Larva

Leg Disc Elongation and Eversion

Adult

Cell shape changes during leg disc elongation

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

a b

Stubble (Sb) encodes a protease that induces changes in cell shape via activation of the RhoA GTPase, resulting in

changes in the actin cytoskeleton

Stubble Mutant6-Hrs. APF

Control 6-Hrs. APF

Cell shape changes that drive leg disc elongation fail in Stubble

mutants

Cell shape changes that drive leg Disc elongation fail in Stubble

mutants

Control6-Hrs. APF

Stubble Mutant6-Hrs. APF

Sb

Leg disc Elongation

Changes in Actin Cytoskeleton

Pastor-Pareja et al. (2004. Dev. Cell 7: 387-399) propose an updated model for imaginal disc eversion. According to their model, imaginal discs evert by

apposing their peripodial side to the larval epidermis, and via invasion of the larval epidermis by cells of the peripodial epithelium and peripodial stalk.

Imaginal Disc Eversion

Normal Leg Development

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The role of how in leg imaginal disc morphogenesis

The Drosophila how gene has pleiotropic functions during

metamorphosis

• how (held-out-wings) also named how, struthio, qkr93F• Encodes KH RNA binding protein • Strong similarity to nematode GLD-1 and mouse QK1• Required for tendon cell differentiation in embryos• how mutants exhibit defects in muscle, muscle attachment,

wing development and adult leg development.

how mutants show defects in leg development

Control how Mutant

how is expressed during metamorphosis

how is expressed in various tissues (including imaginal discs) at the onset of metamorphosis (0-

Hrs. APF)

how Mutants undergo normal cell shape changes that drive leg

imaginal disc elongation

Control 6-Hrs. APF

how Mutant 6-Hrs. APF

how Mutants exhibit defects in leg imaginal disc eversion

Control 6-Hrs. APF

how Mutant 6-Hrs. APF

Control

how Mutant

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Control

how Mutant

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Sb Leg disc Elongation

how Leg disc Eversion

Possible role for how in imaginal disc eversion

According to the Pastor-Pareja et al. model, imaginal discs evert by apposing their peripodial side to the larval epidermis, and via invasion of the larval epidermis by cells of the peripodial epithelium and peripodial stalk.

During this process, the Jun-N-Kinase (JNK) signaling pathway promotes the apposition of peripodial stalk and larval cells, determines the extent of PEMT and motility of the leading edge/peripodial stalk cells, and helps maintain adhesion between larval and imaginal tissue (Pastor-Pareja et al., 2004).

how may play a role in directing interactions between the imaginal disc cells, the cells of the peripodial epithelium and stalk, and larval epithelial cells during disc eversion. Perhaps how regulates expression of genes that play more direct roles in these cell-cell interactions.

The role of ßFTZ-F1 in leg development

Control ßFTZ-F1 Mutant

HypothesisA. ßFTZ-F1, nuclear receptor transcription factor, provides

target genes, including the early genes, BR-C, E74A and E75A, with the competence* to be reinduced by the prepupal ecdysone pulse.

1) These early genes then direct morphogenesis of adult body parts.

B. ßFTZ-F1 provides the prepupal stage-specific E93 early gene with the competence* to be induced by ecdysone.

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

1) E93 then directs programmed cell death in larval body parts.

*Competence the ability to respond to an inductive signal

Morphogenesisof Adult Body Parts

Destruction of Larval body Parts by Programmed Cell Death

Pupariation (Entry into

Metamorphosis)

Evidence in Support of our Hypothesis

• Staining with anti-ßFTZ-F1 antibodies shows ßFTZ-F1 protein bound to the 2B5, 74EF, 75B and 93F puff loci in prepupal salivary gland polytene chromosomes.

• Ectopic expression of ßFTZ-F1 provides E93 with the competence to respond to the late larval ecdysone pulse.

• ßFTZ-F1 protein binds E93 genomic sequences.

• Induction of BR-C, E74A and E75A transcripts by ecdysone is enhanced significantly by ectopic ßFTZ-F1.

• A Loss-of-function mutation in ßFTZ-F1 results in dramatic reductions in E93, E74A, E75A, and BR-C transcripts at the end of the prepupal stage.

• A loss-of-function mutation in ßFTZ-F1 results in pupal lethality with defects in larval salivary gland programmed cell death, head eversion, and leg extension.

Edysone

BR-C

E74A

E75A

E93

ßFTZ-F1

Hours relative to puparium formation

Salivary Gland Developmental Northern Analysis

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

prepupae

ßFTZ-F1 mutants fail to complete metamorphosis

• head eversion

• leg development

• wing development

Mutations in ßFTZ-F1 result in defective leg development

Control ßFTZ-F1 Mutant

ßFTZ-F1 Mutants undergo normal cell shape changes that drive leg imaginal disc elongation

ßFTZ-F1 Mutants undergo normal cell shape changes that drive leg imaginal

disc elongation

Leg extension fails at the prepupal-pupal transition in ßFTZ-F1 mutants

Control

ßFTZ-F1 Mutant

Possible Causes of Short Legs

1) Contraction of the muscles is too weak in ßFTZ-F1 mutants.

2) There is something wrong with the leg imaginal discs in ßFTZ-F1 mutants, which prevents them from extending.

Leg and wing length in ßFTZ-F1 mutants can be rescued by reduced

external pressure

LEGS NMean

LengthStd. Deviation

Std. Error of theMean

Untreated vs. TreatedSig. (2-tailed t-test)

Control Untreated 41 8.89 0.44 0.007

Control Treated 28 9.00 0.65 0.1230.819

Mutant Untreated 27 5.31 0.74 0.144

Mutant Treated 32 6.37 1.65 0.2920.002

WINGS NMean

LengthStd. Deviation

Std. Error of theMean

Untreated vs. TreatedSig. (2-tailed t-test)

Control Untreated 41 7.74 0.43 0.007

Control Treated 28 7.76 0.42 0.0080.441

Mutant Untreated 27 5.17 0.44 0.008

Mutant Treated 32 5.97 1.26 0.2230.002

Table 2. Rescue of Leg and Wing Elongation in ßFTZ-F1 Mutants by a Drop in Pressure

“Untreated” animals were observed at ambient atmospheric temperature.“Treated” animals were subjected to reduced pressure, as described in Materials and Methods.For an explanation of how leg and wing length were recorded, see Materials and Methods.

Significant Difference

Significant Difference

Leg and wing length in ßFTZ-F1 mutants can be rescued by reduced external

pressureControl Untreated vs. Treated

0255075100

10.5109.598.587.576.565.55Denticle Belt Reached by Legs

Percent of Animals

UntreatedTreated

Control Untreated vs. Treated

0255075100

10.5109.598.587.576.565.55Denticle Belt Reached by Wings

Percent of Animals

UntreatedTreated

Mutant Untreated vs. Treated

0255075100

10.5109.598.587.576.565.55Denticle Belt Reached by Legs

Percent of Animals

UntreatedTreated

Mutant Untreated vs. Treated

0255075100

10.5109.598.587.576.565.55Denticle Belt Reached by Wings

Percent of Animals

UntreatedTreated

Possible Causes of Short Legs

1) Contraction of the muscles is too weak in ßFTZ-F1 mutants.

---------------------------------------------------------------2) 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.

This is supported by our careful observations of control and ßFTZ-F1 mutant animals going through pupal ecdysis.

The ßFTZ-F1 mutants exhibit severe defects in the muscle contractions that occur during pupal ecdysis.

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.

ßFTZ-F1 is required for the muscle movements of pupal ecdysis, which generate internal pressure (at the appropriate time), which drives extention of legs and wings, and eversion of the head.

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.

Abdominal muscles

• Play an important role in metamorphosis

• Larval origin but a subset persists after puparium formation to drive morphogenetic events of pupal ecdysis

• Majority of larval muscles are destroyed during prepupal or early pupal stages and replaced by adult structures from muscle precursor cells

Musculature

• Five dorsal muscles• Four lateral transverse

muscles• Segment border

muscles• Ventral muscles

Abdominal Muscle Activity

• Contractions that occur during pupal ecdysis result in:- separation of the pupal cuticle from the puparium (pupal

case)

- shortening of the prepupal body

- translocation of the mid-abdominal gas bubble

- build up of hydrostatic pressure to aid head eversion, leg and wing extension

We have been unable to defects in muscle structure in ßFTZ-F1 mutants (but we are still looking!)

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.

Results

Control 0-Hrs. APF

ßFTZ-F1 Mutant 0-Hrs. APF

ln:longitudinal nerve; sn:segmental nerve; pg:peripheral glia

w; P [w+, Nrv2]; FTZ-F117/DfCat 14hr

w; P [w+, Nrv2]; + 14hr

ln:longitudinal nerve; sn:segmental nerve; pg:peripheral glia

Control 14-Hrs. APF

ßFTZ-F1 Mutant 14-Hrs. APF

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Wild-Type Control

ßFTZ-F1 Mutant

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Recent findings by colleagues have elucidated the role of Ecdysis Triggering Hormone (ETH) in triggering pupal ecdysis.

ETH is a peptide hormone, released into the circulation by specific cells called the INKA cells of the epitracheal gland.

ETH acts on the Central Nervous System, inducing the pupal ecdysis behavioral sequence.

Zitnan, D. et al. J Exp Biol 2003;206:1275-1289

PETH-immunoreactive Inka cells (stained orange/red) in different holometabolous insects

Pupal Ecdysis behavioral sequenceand Leg ExtensionPupariatium

Formation (Entry into

Metamorphosis) Target Genes in

INKA cells

Sbhow

Leg disc ElongationLeg disc Eversion

ETHCNS

FUTURE DIRECTIONShow- We are examining expression patterns of genes in the JNK signaling

pathway in how mutants vs. controls (Blanca Carbajal).

ßFTZ-F1- We are examining the transcription of genes encoding

neuropeptides in ßFTZ-F1 mutants vs. controls (Melanie Ayerh and Kori Matsuura).

- We are continuing to examine motor neurons and muscles in ßFTZ-F1 mutants (Hyowon Choi).

- We are attempting to decipher the molecular mechanism by which ßFTZ-F1 provides target genes with the competence to respond to ecdysone (Antonina Kruppa).

FUTURE DIRECTIONS (cont.)

Other Genes- We are examining Tis-11 mutants, which have abnormal

legs (Hyowon Choi).

- Erika Power is examining genetic interactions between genes involved in leg development.

Acknowledgments

• Mount Holyoke College

• Tina Fortier

• Jennifer McCabe

• Priya Vasa

• Nicole Whyte

• Melanie Ayerh

• Monique Killins

• MANY other MHC independent research students!

• University of Maryland Biotechnology Institute• Eric Baehrecke• Runa Chatterjea• Susan Klinedinst

• Special Thanks for Technical Assistance• Samara Brown• Rachel Fink• Diane Kelly• Stan Rachootin• Marian Rice• Marinko Sremac• Lezek Bledzki

• Funding• National Science Foundation• Howard Hughes Medical Institute• Mount Holyoke College Biology Dept. and

Biochemistry Program

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

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

Morphogenesisof Adult Body Parts

Destruction of Larval body Parts by Programmed Cell Death

Pupariation (Entry into

Metamorphosis) Target Genes?

Cell Death Genes

howSb

Conclusions

• who mutants show multiple lethal phases and pleiotropic effects during metamorphosis.

• who is expressed during metamorphosis.

• who mutant leg discs undergo proper cell shape changes during morphogenesis but do not extend fully at the prepupal-pupal transition.

• This defect is associated with inappropriate orientation of leg imaginal discs.

• The pleiotropic function of who suggests that KH proteins play essential roles in development of numerous cell types.

Acknowledgments

Mount Holyoke College

Tina Fortier

Craig Woodard

University of Maryland Biotechnology Institute

Runa Chatterjee

Susan Klinedinst

Eric Baehrecke