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
QuickTime™ and aCinepak decompressor
are needed to see this picture.
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
QuickTime™ and aCinepak decompressor
are needed to see this picture.
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
QuickTime™ and aVideo decompressor
are needed to see this picture.
Control
how Mutant
how Mutant QuickTime™ and aVideo decompressor
are needed to see this picture.
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
QuickTime™ and aCinepak decompressor
are needed to see this picture.
Wild-Type Control
ßFTZ-F1 Mutant
QuickTime™ and aCinepak decompressor
are needed to see this picture.
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