2025.09.10.675304v1.full-html.html

bioRxiv preprint

developmental time gives larvae exposed to an HSD more time to reach an optimal size. These
results underscore a pivotal role of macrophages in the regulation of

steroid hormone synthesis

under high-calorie dietary regimens, thus positioning these cells as potential therapeutic targets for
the management of metabolic disorders impacting on development.

Results.

A high-sugar diet induces developmental delay and Dpp expression in macrophages

An HSD is defined as a nutritional condition characterized by a significantly elevated sugar
concentration intake relative to a standard diet. Notably, HSDs have been used in

Drosophila

mimic the physiological and developmental alterations observed in humans, including insulin
resistance and chronic inflammation

8,18,22

. Larvae growing under a 10x HSD (4% sucrose in

control diet vs. 40% sucrose in HSD, HSD larvae) showed a ~24-hour (h) developmental delay
compared to those growing in control or standard dietary conditions (control larvae) at the onset
of metamorphosis, also known as the larval-to-pupal transition (Fig. 1A). Exposure to an HSD also
led to a smaller pupal body size (Fig. 1B). The observed negative effect on systemic growth reflects
signs of insulin resistance and resembles the phenotype of mutations in the insulin receptor or
insulin-like peptides

23,24

. To explore whether the observed developmental delay is caused by the

inhibited production of ecdysone—a hormone synthesized in the neuroendocrine prothoracic gland
(PG) that drives the larval-to-pupal transition—we measured the levels of enzymes involved in its
synthesis

, namely

neverland

(

nvl

phantom

(

phm

), and

shadow

(

sad

), at 104 h after egg laying

(AEL), when the onset of pupariation takes place in control larvae. Notably, the mRNA levels of

nvl

phm

, and

sad

at 104 h AEL were significantly reduced in HSD larvae when compared to

control conditions (Fig. 1C). Consistently, the expression of the ecdysone signaling target gene

eip75b

(

e75b

)

was also reduced in HSD larvae (Fig. 1D). Macrophages are ancillary cells

distributed throughout the body and they can sense environmental changes and impact on
surrounding tissues by supplying growth factors and cytokines

16,27,28

. For example, macrophages

in mammals are able to respond to high-sugar and fat diets and are involved in the pathophysiology
of obesity and insulin resistance in adipose tissue

. Similarly,

Drosophila

macrophages can detect

periods of inanition

and modulate ecdysone synthesis and developmental timing

. Given these

considerations, we addressed whether macrophages also play a role in sensing the dietary stress
caused by an HSD and whether they contribute to the regulation of ecdysone synthesis and the
resulting developmental delay. To this end, we used the pan-hemocyte driver

hemolectinΔ

(

hmlΔ

)

to label differentiated macrophages with the fluorescence protein GFP (

hmlΔ>gfp

). Under an HSD,

the  number  of  macrophages  was  reduced  compared  to  controls  (Fig.  1E),  suggesting  that
macrophages respond to dietary stress. To further characterize this response, we carried out bulk
RNA-sequencing of FACS-sorted macrophages of larvae subjected to an HSD and compared their
transcriptional profile to macrophages of control larvae. Broadly, Gene Ontology (GO) analysis
indicated that HSD macrophages showed lower mRNA levels of genes related to carbohydrate
catabolism  and  biosynthesis  (Fig.  S1A).  GO  analysis  also  pointed  to  an  upregulation  of  lipid
metabolism (Fig. S1A and S1B), suggesting that macrophage function relies on triglycerides as an
energy  source,  as  has  been  postulated  in  the  context  of  HSDs

. Moreover, under an HSD,

macrophages surprisingly reoriented towards homeostatic functions instead of classical
immunological ones, a finding supported by the observed upregulation of growth factor-related
GO terms (Fig. S1A, and S1B). Among the secreted proteins whose mRNA levels were
significantly increased in HSD macrophages, we found imaginal disc growth factors (

idgf1

idgf2

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idgf3

idgf4

), the TNFα homolog

egr

, PDGF/VEGF growth factor

pvf2

, the BMP homolog

dpp

the FGF homolog

pyr

, and the growth-blocking factor

gbp3

(Fig. 1F). These observations point to

a potential role of macrophages in sustaining homeostatic functions through inter-organ
communication with distal organs. Of these secreted molecules, our attention was drawn to Dpp,
which has been previously shown to modulate BMP signaling in the PG and to negatively regulate
ecdysone production when overexpressed in distant tissues

31,32

. The expression levels of Brinker

(Brk), a transcriptional repressor negatively regulated by Dpp, were reduced in the macrophages
of HSD larvae (Fig. 1F). The increase in

dpp

levels was confirmed by RT-qPCR, where we used

two housekeeping genes

Rp49

and

Act5C

to avoid the potential dysregulation of metabolic genes

by the HSD (Fig. 1G). We next analyzed whether BMP signaling was activated in the PG of these
larvae. The phosphorylation of the BMP transcription factor Mothers of Dpp (Mad)

and the

absence of Brk expression were used to monitor the activation of BMP signaling

34-36

. pMad levels

were significantly increase

d (

Fig. 1F) and Brk protein levels decreased (Fig. 1G) in the PG of HSD

larvae (Fig. 1G). All these observations point to a potential role of macrophage-derived Dpp in
driving the developmental delay caused by the HSD.

Macrophage-derived Dpp regulates ecdysone synthesis and developmental timing under an
HSD

To test whether macrophage-derived Dpp can regulate developmental timing and ecdysone
synthesis under an HSD, we combined the use of

hmlΔ-Gal4

driver and a

dpp-RNAi

to deplete

dpp

expression specifically in macrophages (Fig. S2A). Pupariation in control larvae took place at ~104
h AEL, whereas HSD larvae pupariated 24 h later (at ~136h AEL). The developmental delay
caused by the HSD was partially rescued by macrophage-specific depletion of

dpp

(Fig. 2A).

Interestingly, the developmental delay caused by the diet was reproduced by overexpressing

dpp

in macrophages of control larvae (Fig. S2B). These data reveal a pivotal role of macrophage-
derived Dpp in delaying developmental timing. To explore whether Dpp regulates developmental
timing by inhibiting ecdysone synthesis in the PG, we next studied the mRNA levels of ecdysone
synthesis enzymes

nvl

phm

, and

sad

, as well as the ecdysone signaling target gene

e75b

in HSD

and control larvae

The increase in the mRNA levels of these three enzymes took place at ~104 h

AEL in control larvae but was delayed by 24 h in HSD larvae and was much milder (Fig. 2B).
Interestingly, macrophage-specific depletion of

dpp

partially restored the peak mRNA levels of

nvl

phm

, and

sad

in HSD larvae, but this increase was still delayed by 24 h compared to control

larvae (Fig. 2B). This observation is consistent with the partial impact of macrophage-specific
depletion of

dpp

on developmental timing (Fig. 2A).

Similar effects on the expression levels of

the ecdysone target gene

e75b

were observed in HSD-larvae upon macrophage-specific depletion

dpp

(Fig. 2C). To corroborate the inhibitory role of macrophage-derived Dpp in ecdysone

synthesis, we next analyzed the total levels of ecdysteroid hormones in HSD and control larvae
upon

dpp

depletion. Again, in HSD larvae, the increase in the ecdysone levels that precedes the

onset of metamorphosis was delayed by 24 h and was much milder (Fig. 2D). Macrophage-specific
depletion of

dpp

also restored the increase in the ecdysone levels, but this increase still occurred

24 h later than in controls (Fig. 2B). Taken together, these data support the notion that macrophage-
derived Dpp inhibits ecdysone synthesis under HSD conditions, thereby modulating
developmental timing. We did not observe macrophages near to or in close contact with the PG
under either physiological or HSD conditions (Fig. S2D). To explore whether macrophage-derived
Dpp can travel from distant macrophages to the PG, we used a transgenic fly expressing a
functional Dpp::GFP fusion protein

. We searched for the presence of GFP fluorescence in the

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PG when Dpp::GFP was specifically expressed in macrophages. Interestingly, the GFP signal was
observed in the PG of these larvae, indicating that macrophage-derived Dpp is secreted and
interacts with PG cells (Fig. 2E). Taken together, these findings indicate that macrophage-derived
Dpp contributes to the HSD-driven developmental delay through inhibition of ecdysone synthesis.

Macrophage-derived Dpp signals to the PG to regulate ecdysone synthesis and developmental
timing under HSD conditions.

The increase in pMad levels observed in the PGs of HSD larvae was rescued upon macrophage-
specific depletion of

dpp

(Fig. 2F), as was the reduction in the levels of Brk (Fig. 2G). These data

support a role of macrophage-derived Dpp in activating BMP signaling in the PG. We next
assessed the functional role of BMP signaling in the PG in regulating ecdysone production and the
developmental delay in HSD larvae. To this end, we combined the use of a PG-specific Gal4-
driver

(phm-GAL4)

and

RNAi

to knock down the Dpp receptor Thickveins (Tkv) and the BMP

signaling transcription factor Mad and analyze the impact of the HSD on developmental timing.
PG-specific depletion of

tkv

fully reverted the HSD-induced increase in pMad levels and the

reduction in Brk levels in the PG (Figure 3A, B). Interestingly, PG depletion of

tkv

and

mad

partially rescued the developmental delay caused by the HSD (Fig. 3C and S3A). Similar results
on developmental timing were obtained upon expression of a dominant negative isoform of Tkv
(Tkv

) in the PG (Fig. S3B)

. These data support the hypothesis that macrophage-derived Dpp

delays

developmental timing through Tkv and Mad in the PG under HSD conditions. To explore

the role of Tkv and Mad in regulating ecdysone production, we measured the mRNA

levels of

ecdysone synthesis enzymes

nvl

phm

, and

sad

, as well as the ecdysone signaling target gene

e75b

HSD larvae upon PG-specific depletion of

tkv

and

mad

. The increase in the

nvl

phm

, and

sad

mRNA levels that occurred in control larvae at ~104 h AEL was delayed by 24 h in HSD larvae,
and this increase was much milder (Fig. 3D and S3C). Interestingly, PG-specific depletion of

tkv

mad

partially restored the mRNA levels of

nvl

phm

, and

sad

in HSD larvae, but this increase

was still delayed by 24 h (Fig. 3D and S3C). This observation is consistent with the partial impact
of the PG-specific depletion of

tkv

mad

on developmental timing

(Fig. 3C and S3A). Similar

effects on the expression levels of the ecdysone target gene

e75b

were observed in HSD-larvae

upon PG-specific depletion of

tkv

mad

(Fig. 3E and S3D). To further confirm the role of BMP

signaling in regulating ecdysone synthesis in the PG, we compared the total levels of ecdysteroid
hormones in HSD and control larvae upon PG-specific depletion of

tkv

. The increase in the

ecdysone levels that precedes the onset of metamorphosis in control larvae was delayed by 24 h
and was much milder in HSD larvae (Fig. 3F). In this regard, PG-specific depletion of

tkv

restored

the increase in ecdysone levels, but this increase still occurred 24 h later than in controls (Fig. 3F).
All these results indicate that macrophage-derived Dpp acts on the PG to inhibit, through Tkv (Fig.
3G) and Mad, ecdysone synthesis and developmental timing in larvae subjected to HSD
conditions.

Dpp-driven developmental delay dampens the negative effects of an HSD on systemic growth

It is well-established that ecdysone can regulate growth in two different ways. First, during early
stages of larval development, ecdysone is present at low levels and interacts with its receptor, EcR,
in somatic tissues to promote proliferative growth

. Second, the ecdysone peak that occurs at the

larval-pupal transition modulates the overall growth period of larvae

Thus, when this peak is

delayed, larvae have more time to grow and display a larger pupal size

. Conversely, when the

peak is induced prematurely, larvae have less time to grow and show a smaller pupal size

. In the

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context of an HSD, the observed reduction in body size is generally thought to be a consequence
of the detrimental effects of the diet on insulin resistance

. We thus questioned whether the effects

of  an  HSD  on  extending  developmental  timing  via  the  production  of  Dpp  by  circulating
macrophages  serve  to  minimize  the  impact  of  insulin  resistance  on  body  growth,  allowing  the
animal to reach a minimal size necessary for adult biological functions. To test this hypothesis, we
analyzed whether partial restoration of developmental timing was able to enhance the negative
impact  of  the  HSD  on  pupal  size.  Interestingly,  macrophage-specific  depletion  of

dpp

, which

partially rescued the developmental delay caused by the HSD (Fig. 2A), further increased the
reduction in pupal size caused by this dietary regimen (Fig. 4A). Similar effects on developmental
timing and pupal size were obtained upon PG-specific depletion of

tkv

mad

(Fig. 4B, C) or

expression of

tkv

(Fig. S3E). We observed that macrophage-specific depletion of

dpp

or PG-

specific depletion of

tkv

mad

in control larvae did not have an impact on pupal size (Fig. 4A).

Interestingly,

dpp

overexpression in the macrophages of control larvae reproduced the effects of

ecdysone inhibition in delaying developmental timing (Fig.  S2B) and increasing pupal size (Fig.
S2C).  Overall,  these  data  support  the  notion  that  the  modulation  of  developmental  timing  by
macrophage-derived  Dpp  under  HSD  conditions  serves  as  a  buffering  mechanism  to  allow  the
organism to extend its developmental period, thereby enabling it to reach a minimum final size
sufficient for adult functions.

Discussion.

Developmental timing, including the onset of puberty, is genetically determined and intricately
mediated by the biosynthesis of sterol hormones

43-47

. However, this finely tuned developmental

program can be significantly modulated by external factors such as tumors and nutritional status

48-

. Recent research has underscored the crucial role of macrophages as pivotal sensor cells capable

of detecting environmental challenges and modulating the physiology of distal organs to maintain
systemic homeostasis

14,16,53-56

. Specifically in mammals, macrophages have been postulated to

stimulate the production of sexual sterol hormones within the gonads

57,58

. In

Drosophila

, the

macrophage genetic cassette

inr/dtor/pvf2

modulates sterol hormone biosynthesis under periods of

inanition during development

. Here, we demonstrate that macrophages also regulate ecdysone

synthesis in

Drosophila

under HSD conditions. Specifically, macrophages secrete the BMP 2/4

homolog  factor  Dpp  to  inhibit  ecdysone  biosynthesis  under  these  dietary  conditions,  thereby
extending developmental timing and allowing the organism to reach an optimal body size. Our
findings  indicate  that  Dpp  interacts  with  the  Tkv  receptor  in  the  PG,  thereby  activating  BMP
signaling and consequently inhibiting the expression of ecdysone biosynthesis enzymes (Fig. 4D).
Here we elucidate the specific physiological conditions and anatomical source from which Dpp is
secreted  and  acts  as  an  inter-organ  signal  to  mediate  ecdysone  synthesis  inhibition  in  the
neuroendocrine  organ,  beyond  its  established  roles  as  a  morphogen  in  the  imaginal  disc

59,60

Furthermore,  this  study  highlights  the  critical  role  of  macrophages,  particularly  macrophage-
secreted  BMPs,  in  mediating  the  progression  of  pathological  conditions  such  as  diet-induced
insulin  resistance.  Notably,  BMP  factors  and  signaling  have  previously  been  implicated  in
promoting  insulin  resistance  and  obesity  in  mice,  and  their  dysregulation  improves  insulin
sensitivity

61-63

. Additionally, these findings have potential implications for understanding the

regulation of sterol hormones across the spectrum of diabetes and insulin resistance in mammals

64-

, offering mechanistic insights into phenomena observed in obese children, where puberty is

often delayed in those with diabetes

67,68

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Materials and methods.

Drosophila husbandry

The plasmatocyte-specific reporter line and driver

hmlΔ-gal4;UAS-2xeGFP

stock was a gift from

S. Sinenko

. Prothoracic gland-specific driver

phm-gal4

was a gift from Michael O’Connor

UAS-tkv

was a gift from Ernesto Sánchez-Herrero,

UAS-dpp::gfp

was a gift from Aurelio

Telemann.

dpp-TRiP.JF02455-RNAi

(36779),

tkv-TRiP.HMS02185-RNAi

(40937),

mad-

TRiP.JF01264-RNAi

(31316),

tkv-lacZ

(11191),

20xUAS-6xmCherry-HA

(52268), and

hmlΔ-gal4

(30139) lines were from the Bloomington Stock Center at Indiana University (Bloomington, IN).
Flies were reared in standard medium (4% glucose, 55 g/L yeast, 0.65% agar, 28 g/L wheat flour,
4 ml/L propionic acid and 1.1 g/L nipagin) at 25°C on a 12-h:12-h light:dark cycle.

For all

experiments, larvae of mixed sexes were used.

Standard diet and 10x high-sugar diet

Strains of

Drosophila melanogaster

and control experiments (control diet) were fed standard

medium (4% glucose, 55 g/L yeast, 0.65% agar, 28 g/L wheat flour, 4 ml/L propionic acid and 1.1
g/L nipagin) at 25°C in 12:12h light- dark cycles. For the high-sugar diet, we added 10x sucrose
to the standard food, increasing the total sugar content to 40%.

Egg-laying and pupariation formation measurements

We crossed 20 males with 20 females. After 48 h, the adult flies were transferred to grape agar
plates (Nutri-fly grape agar premix, Genesee Scientific) supplemented with yeast paste. They were
allowed to lay eggs for 3 to 4 h at 25°C. Following egg deposition, the adults were removed, and
the eggs were incubated at 25°C for an additional 48 h. At this point, second-instar larvae were
collected and transferred to either standard

Drosophila

food (20 larvae per tube) to maintain

physiological conditions or to a 10x high-sugar diet to induce nutritional stress. All larvae were
reared at 25°C. We monitored the larval-to-pupal transition every 8 h, with “time 0” defined as 4
h after the onset of egg laying, referred to as “AEL” (after egg laying).

Pupae volume measurements

Twenty males and twenty females were crossed and allowed to lay eggs over a 24-h period. The
adult flies were transferred to fresh tubes every 24 h, and the deposited eggs were incubated at
25°C. Resulting pupae were collected and photographed with their dorsal side facing upward.
Measurements of length and width were taken using ImageJ, and pupal volume was calculated
using the following formula: v = 4/3π(L/2)(l/2)2 (L, length; and l, width)

. A Leica MZ216F

microscope was used to measure pupal volume, and an Olympus MVX10 macroscope for
representative pupae imaging.

Immunohistochemistry

The brain–ring gland complex was dissected out in cold phosphate-buffered saline (PBS) buffer.
It was then fixed in 4% formaldehyde for 20 min

71,72

and stained with antibodies in PBS with 0.3%

BSA and 0.2% Triton X-100. Next, the brain–ring gland complex was stained overnight at 4°C
with goat anti-GFP (ab6673) (1/500; abcam), mouse anti-Dlg (4F3) (1/50; Hybridoma bank),
rabbit anti-Smad3 (phospho S423 + S425) (C25A9) or pMAD (1/100; Cell Signaling Technology),
chicken

-Gal (ab9361) (1/500; abcam), and guinea pig anti-Brinker (1/100; a gift from Gines

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Morata).  Secondary  antibodies,  Fluorescein  (FITC)  AffiniPure  Donkey  Anti-Goat  IgG  (H+L)
(1/250) (RRID: AB_2340400), Alexa fluor 488-conjugated AffiniPure Donkey Anti-Mouse IgG
(H+L)  (1/250)  (RRID:  AB_2341099),  Alexa  Fluor  488  AffiniPure  Donkey  Anti-Chicken  IgY
(IgG)  (H+L)  (1/250)  (RRID:  AB_2340375),  Cy3-AffiniPure  Donkey  Anti-Mouse  IgG  (H+L)
(1/250)  (RRID:  AB_2315777),  Cy5  AffiniPure  Donkey  Anti-Guinea  Pig  IgG  (H+L)  (1/250)
(RRID:  AB_2340462),  and  Cy3  AffiniPure  Donkey  Anti-Rabbit  IgG  (H+L)  (1/250)
(AB_2307443) were purchased from Jackson ImmunoResearch. For DNA and nuclei staining, we
used DAPI (1/1000). We set up the tile scan with the micrometer-thick Z stack on a Zeiss LSM880
microscope with a 40× oil objective. Images were acquired with a resolution of 1024 × 1024.

Confocal microscopy

Samples were analyzed using a Zeiss LSM 780 confocal microscope. Image acquisition was done
with a 40× oil objective. The most representative image is shown for each experiment.

Flow cytometry analysis of macrophage and macrophage counts

Hemocytes from five larvae at 96 h AEL were bled into 120 μl of FACS buffer (PBS, 0.5% bovine
serum albumin, and 2 mM EDTA) containing 1 nM phenylthiourea (Sigma-Aldrich, St. Louis,
MO, USA) to prevent melanization. We used two forceps to make an incision from the posterior
and pull to the anterior ends of the larvae. We allowed larvae to bleed for a few seconds, and then
carefully scraped the lateral cuticle to avoid the lymph gland disruption

. Macrophages (GFP-

positive hemocytes and 40,6-diamidino-2-phenylindole (DAPI) negative] were extracted from

hmlΔ>2xeGFP

larvae and quantified on a BD FACSARIA III (BD Biosciences). Cells were

selected based on their size with an FSC-A from 2.5K to 250K and their granularity with an SSC-
A from 2.5K to 150K. Later, we gated for singlets (FSC-H vs. FSC-A). For fluorescent hemocytes,
we used a BlueC (530_30) RB-A laser (GFP). Flowcytometry plots were obtained with FlowJo
9.9.

qRT-PCR on sorted macrophages and whole larvae

For qRT-PCR, 15,000 hemocytes were sorted into 600 μl of TRIzol LS (Thermo Fisher Scientific:
10296010).  RNA  extraction  was  performed  following  the  manufacturer’s  instructions  (RNeasy
Mini  Kit,  Qiagen).  RNA  concentration  was  measured  with  a  SpectraMax  QuickDrop  UV-Vis
Spectrophotometer  from  Molecular  Devices.  cDNA  preparation  was  performed  with  the
Invitrogen  SuperScript  IV  First-Strand  Synthesis  System  (Thermo  Fisher  Scientific)  as  per  the
manufacturer’s instructions. qRT-PCR was done with 5 to 10 ng of cDNA.
For whole larvae, qRT-PCR tissue homogenization was performed using a pestle on four to five
larvae per sample, and RNA extraction was performed following the manufacturer’s instructions
(RNeasy Mini Kit, Qiagen). For whole larvae, the same protocol as for the sorted cells was used,
and  the  qRT-PCR  was  performed  on  500  ng  of  cDNA.  qRT-PCR  was  performed  using
QuantStudio6  Flex  Real-Time  PCR  from  Life  Technologies  and  using  TaqMan  Fast  Advance
Mas-termix  and  TaqMan  probes  for

rp49

(Dm02151827_g1),

phm

(Dm01844265_g1),

nvd

(Dm03419116_m1),

sad

(Dm02139323_g1),

eip75b

(Dm01793666_m1), and

dpp

(Dm01842959_m1).

Ecdysteroid measurement

For ecdysteroid extraction, 10 whole larvae from each condition (from 104 h AEL and 128 h AEL,
as indicated in the figure) were collected in 2 ml of tissue-homogenizing mixed beads and then

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frozen in liquid nitrogen and transferred to −80°C. Samples were homogenized using a tissue lyser
in  0.3  ml  of  methanol.  They  were  then  centrifuged  at  maximum  speed  for  5  min  at  room
temperature,  and  the  supernatant  was  transferred  to  another  Eppendorf  tube.  We  repeated  this
procedure by adding 0.3 ml of methanol to the pellet and mixing using vortex, and a third round
using  0.3  ml  of  ethanol.  The  pooled  sample  after  homogenization  was  0.9  ml  per  sample.  For
quantification,  the  extracted  samples  were  centrifuged  at  maximum  speed  for  5  min  (room
temperature) to remove any remaining debris and then divided into two tubes to generate technical
replicates. The cleared samples were evaporated overnight

. The following steps were performed

using the 20-HE enzyme-linked immunosorbent assay kit instructions (Bertin Pharma, no.
A05120; 96 wells). Absorbance was measured at 410 nm with a microplate reader (BioTek
Synergy H1 Plate Reader).

RNA purification, library preparation, and bulk RNA-sequencing

Total RNA from a total of 4 replicates of 20,000 GFP

macrophages was then purified at the IRB

Barcelona Functional Genomics Core Facility, as described in González et al., 2010. mRNA was
isolated from total RNA using the kit NEBNext Poly(A) mRNA Magnetic Isolation Module (New
England Biolabs). NGS libraries were prepared from the purified mRNA using the NEBNext Ultra
II  RNA  Library  Prep  Kit  for  Illumina  (New  England  Biolabs).  Eighteen  cycles  of  PCR
amplification  were  applied  to  all  libraries.  The  final  libraries  were  quantified  using  the  Qubit
dsDNA HS assay (Invitrogen) and quality-controlled with the Bioanalyzer 2100 DNA HS assay
(Agilent). An equimolar pool was prepared with the 20 libraries and submitted for sequencing at
the  Centro  Nacional  de  Análisis  Genómico  (CNAG).  A  final  quality  control  by  qPCR  was
performed by the sequencing provider before paired-end 150 nt sequencing on a NovaSeq6000 S4
(Illumina). More than 275 Gbp of reads were produced, with a minimum of 39 million paired-end
reads per sample.

-Data processing

Adapters

from

reads

were

removed

using

trim

galore

(v.0.6.7)

(https://github.com/FelixKrueger/TrimGalore) with default parameters. Resulting reads were
aligned to the dm6

D. melanogaster

genome version using STAR (v.2.7.10a) (Dobin et al. 2013).

The count matrix was generated in R (https://www.R-project.org/) through the function
“featureCounts” of the Rsubread package (Liao, Smyth, and Shi 2019).
-

Differential expression

Differential expression was measured using the DESeq2 package (Love, Huber, and Anders 2014).
For plotting purposes and generating principal components, the count matrix was normalized using
the “vst” function. The replicate batch was included in the model as a covariate.
-

Gene set enrichment analysis

Gene set enrichment analysis was performed using the roastgsa package (Caballé-Mestres,
Berenguer-Llergo, and Stephan-Otto Attolini 2023). GO and KEGG pathways were downloaded
using the org.Dm.eg.db package.

-Data availability

The raw and processed RNAseq data were deposited in the GEO database under accession number
GSE303750.

Reviewers

can

access

the

data

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE303750 by entering the token
mzgncqwalrkrlyf in the access box.

Statistical analyses

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https://doi.org:10.3390/nu15122749

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All statistical analyses were performed using GraphPad Prism Software 9.0 with a 95% confidence
limit (P < 0.05).

Animal experiments

All animal experiments were done using

Drosophila melanogaster

, which does not require

approval from the Ethics Committee.

Acknowledgments

We  thank  the  Bloomington  Stock  Center  at  Indiana  University  (Bloomington,  IN)  and  the
Developmental Studies Hybridoma Bank (USA) for providing the flies and antibodies. We are
also indebted to the members of the Milán lab for helpful discussion on the manuscript and IRB
Barcelona’s Advanced Digital Microscopy, Functional Genomics and Biostastics/Bioinformatics
Facilities for their assistance. We gratefully acknowledge institutional funding from the Spanish
Ministry of Science, Innovation and Universities through the Centres of Excellence Severo Ochoa
Award, and from the CERCA Programme of the Catalan Government.  M.M.’s lab is funded by a
Spanish Ministry of Science, Innovation and Universities grant. S.J-C was awarded a Beatriu de
Pinós Postdoctoral Fellowship, co-funded by the Marie Skłodowska-Curie program from Horizon
2020  and  the  Agency  for  Management  of  University  and  Research  Grants  of  the  Catalan
Government.

Funding:

S.J-C. Beatriu de Pinós Postdoctoral Fellowship (BP 00177) from the Catalan

Government. M.M., PID2022-137673NB-I00 grants from the Spanish Ministry of Science,
Innovation and Universities.

Author contributions:

S.J-C. conceived the original idea, designed, supervised and performed all

the experiments, contributed to funding acquisition and wrote the original manuscript. M.M.
supervised the project, contributed to funding acquisition, and wrote the original manuscript.

Competing interests:

The authors declare that they have no competing interests.

Data and materials availability:

All data needed to evaluate the conclusions in the paper are

present in the paper and/or the Supplementary Materials. Further information and requests for
resources and reagents should be directed to and will be fulfilled by the Corresponding authors.

References.

Clemente-Suarez, V. J., Beltran-Velasco, A. I., Redondo-Florez, L., Martin-Rodriguez,
A. & Tornero-Aguilera, J. F. Global Impacts of Western Diet and Its Effects on
Metabolism and Health: A Narrative Review.

Nutrients

(2023).

Mayen, A. L., Marques-Vidal, P., Paccaud, F., Bovet, P. & Stringhini, S. Socioeconomic
determinants of dietary patterns in low- and middle-income countries: a systematic
review.

Am J Clin Nutr

100

, 1520-1531 (2014).

https://doi.org:10.3945/ajcn.114.089029

The copyright holder for this preprint

this version posted September 11, 2025.

https://doi.org:10.1146/annurev-nutr-

bioRxiv preprint

Jung, S., Bae, H., Song, W. S. & Jang, C. Dietary Fructose and Fructose-Induced
Pathologies.

Annu Rev Nutr

, 45-66 (2022).

062220-025831

Eckel, R. H., Grundy, S. M. & Zimmet, P. Z. The metabolic syndrome.

Lancet

365

1415-1428 (2005).

https://doi.org:10.1016/S0140-6736(05)66378-7

Lee, J. M.

et al.

Body mass index and timing of pubertal initiation in boys.

Arch Pediatr

Adolesc Med

164

, 139-144 (2010).

https://doi.org:10.1001/archpediatrics.2009.258

Lee, J. M.

et al.

Timing of Puberty in Overweight Versus Obese Boys.

Pediatrics

137

e20150164 (2016).

https://doi.org:10.1542/peds.2015-0164

Starka, L., Hill, M., Pospisilova, H. & Duskova, M. Estradiol, obesity and hypogonadism.

Physiol Res

, S273-S278 (2020).

https://doi.org:10.33549/physiolres.934510

Musselman, L. P.

et al.

A high-sugar diet produces obesity and insulin resistance in wild-

type Drosophila.

Dis Model Mech

, 842-849 (2011).

https://doi.org:10.1242/dmm.007948

Chung, S. Growth and Puberty in Obese Children and Implications of Body Composition.

J Obes Metab Syndr

, 243-250 (2017).

https://doi.org:10.7570/jomes.2017.26.4.243

Li, H.

et al.

Macrophages, Chronic Inflammation, and Insulin Resistance.

Cells

(2022).

https://doi.org:10.3390/cells11193001

Droujinine, I. A.

et al.

Proteomics of protein trafficking by in vivo tissue-specific

labeling.

Nat Commun

, 2382 (2021).

https://doi.org:10.1038/s41467-021-22599-x

Geminard, C., Rulifson, E. J. & Leopold, P. Remote control of insulin secretion by fat
cells in Drosophila.

Cell Metab

, 199-207 (2009).

https://doi.org:10.1016/j.cmet.2009.08.002

Hudry, B.

et al.

Sex Differences in Intestinal Carbohydrate Metabolism Promote Food

Intake and Sperm Maturation.

Cell

178

, 901-918 e916 (2019).

https://doi.org:10.1016/j.cell.2019.07.029

Cox, N.

et al.

Diet-regulated production of PDGFcc by macrophages controls energy

storage.

Science

373

(2021).

https://doi.org:10.1126/science.abe9383

Droujinine, I. A. & Perrimon, N. Interorgan Communication Pathways in Physiology:
Focus on Drosophila.

Annu Rev Genet

, 539-570 (2016).

https://doi.org:10.1146/annurev-genet-121415-122024

Lazarov, T., Juarez-Carreno, S., Cox, N. & Geissmann, F. Physiology and diseases of
tissue-resident macrophages.

Nature

618

, 698-707 (2023).

https://doi.org:10.1038/s41586-023-06002-x

Kubrak, O.

et al.

LGR signaling mediates muscle-adipose tissue crosstalk and protects

against diet-induced insulin resistance.

Nat Commun

, 6126 (2024).

https://doi.org:10.1038/s41467-024-50468-w

Yu, S., Zhang, G. & Jin, L. H. A high-sugar diet affects cellular and humoral immune
responses in Drosophila.

Exp Cell Res

368

, 215-224 (2018).

https://doi.org:10.1016/j.yexcr.2018.04.032

Huang, H.

et al.

Kupffer cell programming by maternal obesity triggers fatty liver

disease.

Nature

(2025).

https://doi.org:10.1038/s41586-025-09190-w

Wang, Z.

et al.

Early life high fructose impairs microglial phagocytosis and

neurodevelopment.

Nature

(2025).

https://doi.org:10.1038/s41586-025-09098-5

The copyright holder for this preprint

this version posted September 11, 2025.

https://doi.org:10.1126/sciadv.adh0589

bioRxiv preprint

Juarez-Carreno, S. & Geissmann, F. The macrophage genetic cassette inr/dtor/pvf2 is a
nutritional status checkpoint for developmental timing.

Sci Adv

, eadh0589 (2023).

Pasco, M. Y. & Leopold, P. High sugar-induced insulin resistance in Drosophila relies on
the lipocalin Neural Lazarillo.

PLoS One

, e36583 (2012).

https://doi.org:10.1371/journal.pone.0036583

Rulifson, E. J., Kim, S. K. & Nusse, R. Ablation of insulin-producing neurons in flies:
growth and diabetic phenotypes.

Science

296

, 1118-1120 (2002).

https://doi.org:10.1126/science.1070058

Shingleton, A. W., Das, J., Vinicius, L. & Stern, D. L. The temporal requirements for
insulin signaling during development in Drosophila.

PLoS Biol

, e289 (2005).

https://doi.org:10.1371/journal.pbio.0030289

Gilbert, L. I., Rybczynski, R. & Warren, J. T. Control and biochemical nature of the
ecdysteroidogenic pathway.

Annu Rev Entomol

, 883-916 (2002).

https://doi.org:10.1146/annurev.ento.47.091201.145302

Segraves, W. A. & Hogness, D. S. The E75 ecdysone-inducible gene responsible for the
75B early puff in Drosophila encodes two new members of the steroid receptor
superfamily.

Genes Dev

, 204-219 (1990).

https://doi.org:10.1101/gad.4.2.204

Mass, E., Nimmerjahn, F., Kierdorf, K. & Schlitzer, A. Tissue-specific macrophages:
how they develop and choreograph tissue biology.

Nat Rev Immunol

, 563-579 (2023).

https://doi.org:10.1038/s41577-023-00848-y

Park, M. D., Silvin, A., Ginhoux, F. & Merad, M. Macrophages in health and disease.

Cell

185

, 4259-4279 (2022).

https://doi.org:10.1016/j.cell.2022.10.007

Lumeng, C. N., Bodzin, J. L. & Saltiel, A. R. Obesity induces a phenotypic switch in
adipose tissue macrophage polarization.

J Clin Invest

117

, 175-184 (2007).

https://doi.org:10.1172/JCI29881

Mahanta, A.

et al.

Macrophage metabolic reprogramming during dietary stress influences

adult body size in Drosophila.

EMBO Rep

(2025).

https://doi.org:10.1038/s44319-025-

00574-7

Denton, D., Xu, T., Dayan, S., Nicolson, S. & Kumar, S. Dpp regulates autophagy-
dependent midgut removal and signals to block ecdysone production.

Cell Death Differ

, 763-778 (2019).

https://doi.org:10.1038/s41418-018-0154-z

Setiawan, L., Pan, X., Woods, A. L., O'Connor, M. B. & Hariharan, I. K. The BMP2/4
ortholog Dpp can function as an inter-organ signal that regulates developmental timing.

Life Sci Alliance

, e201800216 (2018).

https://doi.org:10.26508/lsa.201800216

Kim, J., Johnson, K., Chen, H. J., Carroll, S. & Laughon, A. Drosophila Mad binds to
DNA and directly mediates activation of vestigial by Decapentaplegic.

Nature

388

, 304-

308 (1997).

https://doi.org:10.1038/40906

Campbell, G. & Tomlinson, A. Transducing the Dpp morphogen gradient in the wing of
Drosophila: regulation of Dpp targets by brinker.

Cell

, 553-562 (1999).

https://doi.org:10.1016/s0092-8674(00)80659-5

Jazwinska, A., Kirov, N., Wieschaus, E., Roth, S. & Rushlow, C. The Drosophila gene
brinker reveals a novel mechanism of Dpp target gene regulation.

Cell

, 563-573

(1999).

https://doi.org:10.1016/s0092-8674(00)80660-1

The copyright holder for this preprint

this version posted September 11, 2025.

https://doi.org:10.1038/18451

bioRxiv preprint

Minami, M., Kinoshita, N., Kamoshida, Y., Tanimoto, H. & Tabata, T. brinker is a target
of Dpp in Drosophila that negatively regulates Dpp-dependent genes.

Nature

398

, 242-

246 (1999).

Teleman, A. A. & Cohen, S. M. Dpp gradient formation in the Drosophila wing imaginal
disc.

Cell

103

, 971-980 (2000).

https://doi.org:10.1016/s0092-8674(00)00199-9

Haerry, T. E., Khalsa, O., O'Connor, M. B. & Wharton, K. A. Synergistic signaling by
two BMP ligands through the SAX and TKV receptors controls wing growth and
patterning in Drosophila.

Development

125

, 3977-3987 (1998).

https://doi.org:10.1242/dev.125.20.3977

Perez-Mockus, G.

et al.

The Drosophila ecdysone receptor promotes or suppresses

proliferation according to ligand level.

Dev Cell

, 2128-2139 e2124 (2023).

https://doi.org:10.1016/j.devcel.2023.08.032

Delanoue, R., Slaidina, M. & Leopold, P. The steroid hormone ecdysone controls
systemic growth by repressing dMyc function in Drosophila fat cells.

Dev Cell

, 1012-

1021 (2010).

https://doi.org:10.1016/j.devcel.2010.05.007

McBrayer, Z.

et al.

Prothoracicotropic hormone regulates developmental timing and body

size in Drosophila.

Dev Cell

, 857-871 (2007).

https://doi.org:10.1016/j.devcel.2007.11.003

Rewitz, K. F., Yamanaka, N., Gilbert, L. I. & O'Connor, M. B. The insect neuropeptide
PTTH activates receptor tyrosine kinase torso to initiate metamorphosis.

Science

326

1403-1405 (2009).

https://doi.org:10.1126/science.1176450

Danielsen, E. T., Moeller, M. E. & Rewitz, K. F. Nutrient signaling and developmental
timing of maturation.

Curr Top Dev Biol

105

, 37-67 (2013).

https://doi.org:10.1016/B978-0-12-396968-2.00002-6

Plant, T. M. Neuroendocrine control of the onset of puberty.

Front Neuroendocrinol

73-88 (2015).

https://doi.org:10.1016/j.yfrne.2015.04.002

Bordini, B. & Rosenfield, R. L. Normal pubertal development: Part I: The endocrine
basis of puberty.

Pediatr Rev

, 223-229 (2011).

https://doi.org:10.1542/pir.32-6-223

Veldhuis, J. D.

et al.

Estrogen and testosterone, but not a nonaromatizable androgen,

direct network integration of the hypothalamo-somatotrope (growth hormone)-insulin-
like growth factor I axis in the human: evidence from pubertal pathophysiology and sex-
steroid hormone replacement.

J Clin Endocrinol Metab

, 3414-3420 (1997).

https://doi.org:10.1210/jcem.82.10.4317

Yamanaka, N., Rewitz, K. F. & O'Connor, M. B. Ecdysone control of developmental
transitions: lessons from Drosophila research.

Annu Rev Entomol

, 497-516 (2013).

https://doi.org:10.1146/annurev-ento-120811-153608

Ohhara, Y., Kobayashi, S. & Yamanaka, N. Nutrient-Dependent Endocycling in
Steroidogenic Tissue Dictates Timing of Metamorphosis in Drosophila melanogaster.

PLoS Genet

, e1006583 (2017).

https://doi.org:10.1371/journal.pgen.1006583

Villamor, E. & Jansen, E. C. Nutritional Determinants of the Timing of Puberty.

Annu

Rev Public Health

, 33-46 (2016).

https://doi.org:10.1146/annurev-publhealth-031914-

122606

Romao, D., Muzzopappa, M., Barrio, L. & Milan, M. The Upd3 cytokine couples
inflammation to maturation defects in Drosophila.

Curr Biol

, 1780-1787 e1786

(2021).

https://doi.org:10.1016/j.cub.2021.01.080

The copyright holder for this preprint

this version posted September 11, 2025.

https://doi.org:10.1126/science.1216689

bioRxiv preprint

Colombani, J., Andersen, D. S. & Leopold, P. Secreted peptide Dilp8 coordinates
Drosophila tissue growth with developmental timing.

Science

336

, 582-585 (2012).

Garelli, A., Gontijo, A. M., Miguela, V., Caparros, E. & Dominguez, M. Imaginal discs
secrete insulin-like peptide 8 to mediate plasticity of growth and maturation.

Science

336

579-582 (2012).

https://doi.org:10.1126/science.1216735

Woodcock, K. J.

et al.

Macrophage-derived upd3 cytokine causes impaired glucose

homeostasis and reduced lifespan in Drosophila fed a lipid-rich diet.

Immunity

, 133-

144 (2015).

https://doi.org:10.1016/j.immuni.2014.12.023

Hersperger, F.

et al.

DNA damage signaling in Drosophila macrophages modulates

systemic cytokine levels in response to oxidative stress.

Elife

(2024).

https://doi.org:10.7554/eLife.86700

Ayyaz, A., Li, H. & Jasper, H. Haemocytes control stem cell activity in the Drosophila
intestine.

Nat Cell Biol

, 736-748 (2015).

https://doi.org:10.1038/ncb3174

Sriskanthadevan-Pirahas, S., Tinwala, A. Q., Turingan, M. J., Khan, S. & Grewal, S. S.
Mitochondrial metabolism in Drosophila macrophage-like cells regulates body growth
via modulation of cytokine and insulin signaling.

Biol Open

(2023).

https://doi.org:10.1242/bio.059968

Lombard-Vignon, N., Grizard, G. & Boucher, D. Influence of rat testicular macrophages
on Leydig cell function in vitro.

Int J Androl

, 144-159 (1992).

https://doi.org:10.1111/j.1365-2605.1992.tb01123.x

Afane, M.

et al.

Modulation of Leydig cell testosterone production by secretory products

of macrophages.

Andrologia

, 71-78 (1998).

https://doi.org:10.1111/j.1439-

0272.1998.tb01149.x

Nellen, D., Burke, R., Struhl, G. & Basler, K. Direct and long-range action of a DPP
morphogen gradient.

Cell

, 357-368 (1996).

https://doi.org:10.1016/s0092-

8674(00)81114-9

Lecuit, T.

et al.

Two distinct mechanisms for long-range patterning by Decapentaplegic

in the Drosophila wing.

Nature

381

, 387-393 (1996).

https://doi.org:10.1038/381387a0

Baboota, R. K., Bluher, M. & Smith, U. Emerging Role of Bone Morphogenetic Protein 4
in Metabolic Disorders.

Diabetes

, 303-312 (2021).

https://doi.org:10.2337/db20-0884

Son, J. W.

et al.

Association of serum bone morphogenetic protein 4 levels with obesity

and metabolic syndrome in non-diabetic individuals.

Endocr J

, 39-46 (2011).

https://doi.org:10.1507/endocrj.k10e-248

Schulz, T. J.

et al.

Loss of BMP receptor type 1A in murine adipose tissue attenuates age-

related onset of insulin resistance.

Diabetologia

, 1769-1777 (2016).

https://doi.org:10.1007/s00125-016-3990-8

Beaudry, J. L., D'Souza A, M., Teich, T., Tsushima, R. & Riddell, M. C. Exogenous
glucocorticoids and a high-fat diet cause severe hyperglycemia and hyperinsulinemia and
limit islet glucose responsiveness in young male Sprague-Dawley rats.

Endocrinology

154

, 3197-3208 (2013).

https://doi.org:10.1210/en.2012-2114

Davani, B.

et al.

Aged transgenic mice with increased glucocorticoid sensitivity in

pancreatic beta-cells develop diabetes.

Diabetes

53 Suppl 1

, S51-59 (2004).

https://doi.org:10.2337/diabetes.53.2007.s51

Gyawali, P.

et al.

The role of sex hormone-binding globulin (SHBG), testosterone, and

other sex steroids, on the development of type 2 diabetes in a cohort of community-

The copyright holder for this preprint

this version posted September 11, 2025.

https://doi.org:10.1007/s00592-018-1163-6

bioRxiv preprint

dwelling middle-aged to elderly men.

Acta Diabetol

, 861-872 (2018).

Ghitha, N., Vathania, N., Wiyono, L. & Pulungan, A. Delayed menarche in children and
adolescents with type 1 diabetes mellitus: a systematic review and meta-analysis.

Clin

Pediatr Endocrinol

, 104-112 (2024).

https://doi.org:10.1297/cpe.2023-0058

Rohrer, T.

et al.

Delayed pubertal onset and development in German children and

adolescents with type 1 diabetes: cross-sectional analysis of recent data from the DPV
diabetes documentation and quality management system.

Eur J Endocrinol

157

, 647-653

(2007).

https://doi.org:10.1530/EJE-07-0150

Sinenko, S. A. & Mathey-Prevot, B. Increased expression of Drosophila tetraspanin,
Tsp68C, suppresses the abnormal proliferation of ytr-deficient and Ras/Raf-activated
hemocytes.

Oncogene

, 9120-9128 (2004).

https://doi.org:10.1038/sj.onc.1208156

Colombani, J.

et al.

A nutrient sensor mechanism controls Drosophila growth.

Cell

114

739-749 (2003).

https://doi.org:10.1016/s0092-8674(03)00713-x

Juarez-Carreno, S.

et al.

Body-fat sensor triggers ribosome maturation in the

steroidogenic gland to initiate sexual maturation in Drosophila.

Cell Rep

, 109830

(2021).

https://doi.org:10.1016/j.celrep.2021.109830

Palomino-Schatzlein, M., Carranza-Valencia, J., Guirado, J., Juarez-Carreno, S. &
Morante, J. A toolbox to study metabolic status of Drosophila melanogaster larvae.

STAR

Protoc

, 101195 (2022).

https://doi.org:10.1016/j.xpro.2022.101195

Ramond, E.

et al.

The adipokine NimrodB5 regulates peripheral hematopoiesis in

Drosophila.

FEBS J

287

, 3399-3426 (2020).

https://doi.org:10.1111/febs.15237

Texada, M. J.

et al.

Autophagy-Mediated Cholesterol Trafficking Controls Steroid

Production.

Dev Cell

, 659-671 e654 (2019).

https://doi.org:10.1016/j.devcel.2019.01.007

The copyright holder for this preprint

this version posted September 11, 2025.

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macrophages (

hmlΔ>gfp

) of larvae growing in control diet and HSD. n=3. (F) Volcano plot

illustrating  the  differential  gene  expression  in  macrophages.  These  macrophages  were  isolated
from larvae exposed to either a control diet or HSD (n=4). Red indicates upregulation and blue
indicates  downregulation  of  gene  expression  under  the  HSD  condition  (adjusted  P<0.05;  |fold
change|≥1.5). Relevant growth factor genes are labeled with their corresponding gene symbols.
Fold  changes  and  p-values  were  calculated  using  the  DESeq2  R  package.  (G)  Transcriptional
levels of

dpp

in larvae growing under control diet and HSD. mRNA levels were normalized to

rp49

and

Act5C

. Samples were pooled by five larvae per condition. n=4. (H, I) pMad (H) and Brk

(I) protein expression levels in prothoracic glands of 96 h AEL-old larvae growing in control diet
and HSD. Images show a Z-stack and scale bars of 10

m. n

6. n

6. Values represent the means

± SD. Data were analyzed by two-tailed unpaired t test, and. *P < 0.05, **P < 0.01, ***P < 0.001,
and ****P < 0.0001.

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Figure 2. Macrophage-derived Dpp signals to the prothoracic gland to inhibit ecdysone
synthesis and to cause a developmental delay under high-sugar diet.

(A) Developmental timing

in larvae of the indicated genotypes growing in control diet or HSD (n=3, 20 larvae per genotype
in three independent crosses, 60 larvae in total per condition and genotype). (B, C) Transcriptional
levels of the ecdysone biosynthesis enzymes

nvl

phm

, and

sad

(B) and the ecdysone signaling

target gene,

e75b

mRNA levels were normalized to

rp49

. n=4. (D) Total ecdysteroid levels in the indicated

genotypes and conditions and quantified by ELISA at the indicated time points. n = 4, with each
sample pooled from 10 larvae. (E) Macrophages can secrete a Dpp tagged with GFP, which can
be detected in the prothoracic gland. n

5. (F, G) pMad (F) and Brk (G) protein expression levels

in the prothoracic gland at 96 h AEL in the indicated genotypes and conditions. Images in E-G
show a Z-stack and scale bars of 10

m. F: n

7. G: n

6. Values represent the means ± SD. Data

in B, C, and D were analyzed by two-tailed unpaired t test, and *P < 0.05, and **P < 0.01. Data in
F and G were analyzed by non-parametric Kruskal–Wallis test and multiple comparison and ****P
< 0.0001.

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Figure 3. Dpp signaling in the prothoracic gland inhibits ecdysone synthesis and induces a
developmental delay under a high-sugar diet

(A, B) pMad (A) and Brk (B) protein expression

levels in the prothoracic gland at 96 h AEL in the indicated genotypes and conditions. n

5. (C)

Developmental timing in larvae of the indicated genotypes growing in control diet or HSD (n=3,
20 larvae per genotype in three independent crosses, 60 larvae in total per condition). (D, E)
Transcriptional levels of the ecdysone biosynthesis enzymes

nvl

phm

, and

sad

(D) and the

ecdysone signaling target gene,

e75b

(E) measured across the indicated conditions, genotypes and

time points. mRNA levels normalized to

rp49

. n=4. (F) Total ecdysteroid levels in the indicated

genotypes and conditions, quantified by ELISA at the indicated time points. n = 4, with each
sample pooled from 10 larvae. (G) Tkv expression in the prothoracic gland. Images in A, B, G
show a Z-projection and scale bars of 10

m (A, B) or 30

m (G). Values represent the means ±

SD. Data were analyzed in D, E, and F by two-tailed unpaired t test, and *P < 0.05, **P < 0.01,
***P < 0.001, and ****P < 0.0001. Data were analyzed in A and B by non-parametric Kruskal–
Wallis test and multiple comparison and ****P < 0.0001.

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Figure S2, related to Fig. 2. Macrophages overexpressing Dpp induce a developmental delay
and drive systemic growth.

(A) RNAi efficiency of

dpp

RNAi line. mRNA levels were

normalized to

rp49

at 96 h AEL of sorted macrophages in the indicated genotypes. n=4, with each

sample pooled from 15,000 macrophages. (B) Developmental timing in larvae of the indicated
genotypes and growing in control diet (n=3, 20 larvae per genotype in three independent crosses,
60 larvae in total per condition). (C) Final pupal size of larvae overexpressing

dpp

specifically in

macrophages and growing in control diet. n=35. (D) Representative images of the prothoracic
gland from larvae subjected to a physiological diet or 10x high-sugar diet. Macrophages are labeled
with GFP fluorescent protein (

hml

>gfp)

. Images show a Z-stack and scale bars of 30

m. n=6.

Data were analyzed by two-tailed unpaired t test, and values represent the means ± SD. *P < 0.05,
and ***P < 0.001.

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Fig. S3, related to Fig.3 and Fig.4. A role of Dpp signaling in inhibiting ecdysone synthesis in
the prothoracic gland and inducing a developmental delay under a high-sugar diet.

(A, B)

Developmental timing in larvae of the indicated genotypes and growing in control diet or HSD
(n=3, 20 larvae per genotype in three independent crosses, 60 larvae in total per condition). (C, D)
Transcriptional levels of the ecdysone biosynthesis enzymes,

nvl

phm

, and

sad

ecdysone signaling target gene,

e75b

(D) were quantified across the indicated genotypes,

conditions and time points. mRNA levels normalized to

rp49

. n=4. (E) Final pupal size of larvae

growing in control diet or HSD and subjected to macrophage-specific expression of a dominant
negative version of the Tkv receptor (Tkv-DN). n

30. Data were analyzed by two-tailed unpaired

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