Supplemental Information
gene name
forward
reverse
reference
Clock
TCAGGCCCCACGTCTTTTAC
GCCGCCTGGAGGATCAG
Kopp et al., 2010
sirt1
ACAGAGCCATGAAGCAGGAT
GAGGCACGTCATGAGGAATC
this study
per1
CCGTCAGTTTCGCTTTTCTC
ATGTGCAGGCTGTAGATCCC
Cavallari et al., 2011
per2
ATGTCGATGGCTTTAGGCAG
CGAGACATCCAGAAGGTGCT
Cavallari et al., 2011
cry1a
TCCGCTGTGTGTACATCCTC
CAAACACTGCAGCAAAAACC
Cavallari et al., 2011
cry2a
GGACCAATACACCAGCACCAG
CAGCAAGTGTCCTGCCATGTC
Amaral et al., 2011
rev-erbα
TGCTTAGCAGTTATGGCTTTATCG
GCAGGGTTTTGACAGAATTTCC
this study
pparαa
ATTATGTACAGCCCTCTGAGCGGA
TGAGAACACTTCTGAGGACGGACT
Tseng et al., 2011
pparβb
CGTCAACACAGCCTACCTGA
GCCACAGGGAGTCCATATCA
this study
pparγ
CATACACAAGAAGAGCCGCA
ATGTGGTTCACGTCACTGGA
this study
Agrp
GTCCACCTGCAGAGAAGAGG
AGAAGGCCTTAAAGAAGCGG
Zhang et al., 2010
Npy
CCAAACATGAAGATGTGGATGAG
CCAAGCAGACGAACAAGAGAAA
this study
Lep
GCAAAATTTACTTCCAA
CGCTTTCCCATTTGTTGATT
this study
Lpl
GGCCAAATTTGTCAACTGGT
CATGAGCGCCAAGACTGTAA
this study
SI 1: Primer sequences. Specific forward and reverse primer for qRT-PCR were developed or
adopted from literature.
SI 2: mRNA expression of hormone and neurotransmitters. (a-c) Relative mRNA expression was
quantified by qRT-PCR. Samples were taken at 15 dpf at two different ZT to show alterations in
mRNA expression due to circadian rhythms. Values are shown as mean ± SE and significance is
marked with *.
SI 3: Regression analysis of gene expression of zebrafish larvae raised under control or
continuous light (LL) conditions. (a) Ppparβδ and reverbα mRNA expression is highly
correlated in zebrafish raised under LL but not control light conditions. (b) Overview of
regression analysis of individual genes. Only genes are shown in which regression analysis
revealed R² ≥ 0.95. Genes compared are shown with a line. Grey lines indicate a relation under
control conditions; black lines indicate a relation under LL conditions. For example, under LL
conditions leptin mRNA expression was highly correlated to sirt1, pparβδ, lpl, rev-erbα and
per1.
SI 4: Adipocytes and lipid profile. (a) Volumes of adipocyte lipid droplets based on methylene
signal per fish. Values are shown as mean ± SE. (b) Mean volumes of adipocyte lipid droplets
based on methylene signal per fish. Values are shown as mean ± SE. (c) Fatty acid and (d)
triglyceride content of pooled and homogenized 15 dpf old larvae zebrafish larvae. Values were
normalized to sample DNA content. Values are shown as mean ± SE and significance is marked
with *. (e) Lipid profile of 15 dpf old zebrafish larvae under different treatment conditions. Red
marks increased values, green marks decreased values [ng / µg DNA].
right
eye
left
eye
pineal gland
SI 5: Rev-erbα and Pparγ protein expression. (a) Western blot against Rev-erbα (molecular
weight: 69.46 kDa) using 100 µg total protein of ten 25 dpf old zebrafish larvae raised under
control conditions. (b) Immunohistochemical staining against Rev-erbα (red; Alexa 568 goat anti
rabbit) and Pparγ (green; Alexa 488 goat anti mouse) of the zebrafish head region revealed a
clear Rev-erbα signal in the pineal gland. (c) Immunohistochemical staining against Rev-erbα
and Pparγ of control zebrafish showing the region where adipocytes should develop. No
adipocytes and no Pparγ signal were detected. (d) Immunohistochemical staining against Reverbα and Pparγ of control zebrafish showing muscle tissue. Rev-erbα and Pparγ signal colocalized. Marked area was zoomed in (e).
SI 6: Survival, activity and size. (a) Survival and (b) activity were monitored over the whole
experimental period. (c) DNA content of pooled 15 dpf old zebrafish larvae was quantified by a
photospectrometer.
SI 7: Transcription Factor Binding Site Analysis (ConTra). Using the online tool ConTra (1) 500
bp promoter regions of zebrafish ppar isoforms (pparα NM_001161333, pparβδ NM_131468 and
pparγ NM_131467) were analyzed for Rev-erbα binding sites (REV-RE: A/T GGTCA). Pparα
promoter region (500 bp) comprised no REV-REs while pparβδ promoter included three REVREs at position -129, -118 and -77 and pparγ promoter one REV-REs at position -366. No Eboxes for Clock/Bmal binding were found. REV-REs were marked with bold capital letters.