Ecdysone receptor expression in the CNS correlates

with stage-specific responses to ecdysteroids during

Drosophilia and Manduca development

Truman, et al. (1994)

 Introduction
Ecdysteroids produced by Ecdysone receptor (EcR) and ultraspiracle (USP) heterodimer transcription factor
regulate CNS transformation from larval to adult form in Drosophila melanogaster and Manduca sexta
EcR-A, EcR-B1, and EcR-B2
expression regulates CNS at different periods of development
 Developmental changes in Drosophila Life CNS EcR-A
expressed by neurons during pupal-adult
EcR-B1 transformation
high levels in larval neurons at start of metamorphosis
larval features lost in response to ecdysteroids Imaginal Neurons
correlated with proliferative activity or regressive responses adult-specific
imaginal neurons
during larval
generated during
molts: no
larval stage, but do
neuronal
not appear until
receptors and no
metamorphosis
response to
ecdysteroids many imaginal
neurons produce
information
processing, learning,
EcR-B2
and behavior
at time of this study
associated brain
(1994) no antibody
structures (e.g.
specific to this
mushroom bodies)
isoform had been
obtained
2015: loss of
function isoform
effects on cryptic
unstable transcripts
 Hypothesis

Neurons in Drosophila melanogaster and Manduca sexta exhibit qualitative and

quantitative changes in ecdysone receptor expression throughout their life cycle and
the differences correlate with specific patterns of ecdysteroid response
 Materials and Methods

Experimental animals
Drosophila
Canton-S wild-type
raised at 25C on standard medium
eggs collected on agar plates with yeast paste
nervous systems examined at 6 to 8hr intervals throughout larval life

Manduca
larvae raised at 26C on artificial diet

Over 1,700 nervous systems extracted from Drosophila and Manduca

 Materials and Methods
Antibodies and Immunohistochemistry
Drosophila
antibodies directed against epitopes in common region of EcR isoforms
rabbit polyclonal antibodies (mAbs)
monoclonal antibodies
EcR-A-specific
rabbit polyclonal antiserum raised against EcR-A-specific fusion protein and mAbs 15G1a, 18F6,
and 12H4
EcR-B1-specific
mAb AD4.4
biotinylated secondary antibodies
EcR detected via fluorescence or enzyme-linked detection
Manduca
polyclonal anti-EcR and mAb JG6.2
preblocking for anti-EcR performed with 1:1000 dilution of affinity-purified antibody and various
concentrations of TrpE-EcR fusion protein and control TrpE protein
EcR detected via fluorescence or enzyme-linked detection
 Materials and Methods

Quantification of immunosignal
scanning confocal microscopy used to determine cell type and select nuclei for measurement
Area program use to measure avg. pixel intensity in each nucleus

Combined autoradiography and receptor immunocytochemistry

thaw mounted onto slides
photodeveloped for 6mins in Kodak D-170 developer at 16C
immunostained with nickel sulfate-diaminobenzidine intensification procedure
stained with chromagen
no staining observed when immune reagents were omitted
 Results
Distinct patterns of immunostaining observed when CNSs stained with EcR-A and EcR-B1
antibodies
Fig. 5.
immunostained ventral CNS from Drosophila during
first half of metamorphosis showing distribution of
staining
A,B: white puparium stage
C,D: 25hrs after puparium formation (APF)
small arrowhead: midline glia
large arrowhead: perineuropilar glia
arrow: type II larval neurons
E,F: 55 hrs APF
arrows: type II neruon
m: muscle
a: abdominal neuromeres
t: thoracicd neuromeres
after puparim formation (APF)
 Results
hrs APF
White
puparium
stage: weak EcR-A
staining in
prominent larval
EcR-B1 neurons
staining in
larval
neurons

loss of lamina glia

Ecr-B1 in (arrow) and
neurons, ring gland
but (arrowhead)
prominent are positive
tracheal for EcR-A
staining
(arrow)
 Results

hrs APF

prominent EcR-A staining in perineuropilar glia

 Results
a nuclear antigen in Manduca was recognized by two mAbs (JG6.2 and GGD11.6) directed against epitopes in the C
region of Drosophila EcR and by polyclonal antiserum raised against the D region
this supports previous findings of 95% similarity in C regions between Drosophila and Manduca
likely that Manduca EcR is being recognized by the Drosphila antibodies

antibody preblocked with TrpE-EcR fusion

protein

antibody preblocked with 4x higher

concentration of control Trpe protein
larval neurons show nuclear Ecr staining (open
arrow) but immature, imaginal neurons (arrow)
are negative
 Results
antibodies that reacted with Manduca EcR recognize common regions of the receptor
provides data re: changes in total levels of receptor, but not which isoforms are responsible for changes

neurons immunopositive for mAb JG6.2 exhibited

nuclear binding of 125I-ponasterone, evidenced by
silver grains
clusters of imaginal neurons did not exhibit nuclear
binding
colocalization of immunostaining and 125I-ponasterone
binding indicate that Manduca EcR detected by
antibodies is part of an active receptor complex
 fluorescent signal often found in cytoplasm remained when primary antibody omitted or was preabsorbed with
EcR protein
likely due to cytoplasmic autofluorescence
consistent with evidence showing accumulation of significant radiolabeled ecdysteroids only in nuclei of
Manduca neurons
 Time course of EcR expression in larval neurons
Drosophila immunostained with mAb AD4.4 to show EcR-B1

(A) 10hr 1st instar larva (34 hrs after egg laying (AEL); arrowheads: pair of weakly staining, brain neurons
(B) 12hrs 2nd instar larva (60 hrs AEL); weak staining in tracheal nuclei (arrow); mouth hooks (mh) show strong nuclear
staining
(C-E) 3rd instar larvae at 14 (C, 86hrs AEL), 30 (D, 102hrs AEL) and 40 (E, 112hrs AEL) hours postecdysis
(C)As larvae aged, B1 staining was lost from OL proliferation zones (O), but appeared in larval neurons (D) and
mushroom body neurons (E; m)
 Relative levels of immunofluorescence due to EcR-A (open bars) and EcR-B1 (black bars) in
nuclei in Manduca
(A) isolated neuroblasts in central brain and thoracic ganglia
(B) imaginal neurons in ventral throacic area
(C) superficial neurons of mushcroom bodies
age-related shift in EcR expression
1st no receptors, then EcR-B1 and finally
EcR-A
Expression of EcR isoforms in glial cells in Drosophila
black bars: EcR-B1
white bars: EcR-A
dashed boxes: periods of weak expression
 Discussion

Type I pattern (most cells)

last instar: high EcR-B1
metamorphosis: B1 isoform lost, only EcR-A remains
end of metamorphosis: ecdysteroid surge causes formation of adult with sprouting and synaptogenesis

Type II pattern
metamorphosis: EcR-A exceptionally high in some instances
programmed death after adult emergence

Type III
pupariation: no EcR-B1
 Discussion
Bryant, et al. (2015): EcR-B2
loss of function (dominant negative) isoforms decrease Cut expression in dorsal-ventral boundary
 Discussion

What is responsible for EcR dynamics?

A and B1 isoforms encoded by overlapping transcription units with different promoters and can be separately
controlled
B1 and B2 isoforms encoded by mRNAs derived from EcR-B primary transcript by alternate splicing

juvenile hormone (JH)

This study: 1994
Manduca: enhanced neuronal expression of EcR in final instar occurs after normal decline in JH
Drosophila: JH responses are poor and cannot currently be tested
Takumi, et al. (2017)
JH represses ecdysone-induced protein 93F (E93)
prevents immature larvae from bypassing pupal stage and progressing to adult development
JH-inducible Kruppel homolog 1 is transcriptional repressor of E93

ecdysteroids themselves initiating expression of specific EcRs