of 29
Barlow
et al
. eLife 2023;12:RP87521. DOI: https://doi.org/10.7554/eLife.87521
1 of 29
The zebrafish mutant
dreammist
implicates sodium homeostasis in
sleep regulation
Ida L Barlow
1†
, Eirinn Mackay
1‡
, Emily Wheater
, Aimee Goel
1
, Sumi Lim
1
,
Steve Zimmerman
2
, Ian Woods
3
, David A Prober
4
, Jason Rihel
1
*
1
Department of Cell and Developmental Biology, University College London, London,
United Kingdom;
2
Department of Molecular and Cellular Biology, Harvard University,
Cambridge, United States;
3
Ithaca College, New York, United States;
4
Division of
Biology and Biological Engineering, California Institute of Technology, Pasadena,
United States
Abstract
Sleep is a nearly universal feature of animal behaviour, yet many of the molec-
ular, genetic, and neuronal substrates that orchestrate sleep/wake transitions lie undiscovered.
Employing a viral insertion sleep screen in larval zebrafish, we identified a novel gene,
dreammist
(
dmist
), whose loss results in behavioural hyperactivity and reduced sleep at night. The neuronally
expressed
dmist
gene is conserved across vertebrates and encodes a small single-
pass transmem-
brane protein that is structurally similar to the Na
+
,K
+
- ATPase regulator, FXYD1/Phospholemman.
Disruption of either
fxyd1
or
atp1a3a
, a Na
+
,K
+
- ATPase alpha-
3 subunit associated with several
heritable movement disorders in humans, led to decreased night-
time sleep. Since
atpa1a3a
and
dmist
mutants have elevated intracellular Na
+
levels and non-
additive effects on sleep amount at
night, we propose that Dmist-
dependent enhancement of Na
+
pump function modulates neuronal
excitability to maintain normal sleep behaviour.
eLife assessment
This study offers new
fundamental
information on a role for the sodium/potassium pump in sleep
regulation. Elegant methods were used to provide
compelling
evidence supporting the claim.
The work will be of interest to sleep researchers in zebrafish as well as in other species for future
investigation.
Introduction
The ability of animals to switch between behaviourally alert and quiescent states is conserved across
the animal kingdom (
Cirelli, 2009
;
Joiner, 2016
). Fundamental processes that govern the regulation
of sleep-
like states are shared across species, such as the roles of circadian and homeostatic cues in
regulating the time and amount of sleep, stereotyped postures, heightened arousal thresholds, and
the rapid reversibility to a more alert state (
Joiner, 2016
). The near ubiquity of sleep implies that it
serves ancient functions and is subject to conserved regulatory processes. However, many key molec-
ular components that modulate sleep and wake states remain undiscovered.
Over the past two decades, investigations into sleep and arousal states of genetically tractable
model organisms, such as
Drosophila melanogaster
,
Caenorhabditis elegans
, and
Danio rerio
(zebrafish), have uncovered novel molecular and neuronal components of sleep regulation through
gain- and loss-
of-
function genetic screens (reviewed in
Barlow and Rihel, 2017
;
Sehgal and Mignot,
RESEARCH ARTICLE
*For correspondence:
j.rihel@ucl.ac.uk
Present address:
MRC London
Institute for Medical Sciences,
Imperial College, London,
United Kingdom;
Sainsbury
Wellcome Centre for Neural
Circuits and Behaviour, University
College London, London, United
Kingdom;
§
MRC Centre for
Reproductive Health, University
of Edinburgh, Edinburgh, United
Kingdom
Competing interest:
The authors
declare that no competing
interests exist.
Funding:
See page 23
Preprint posted
05 March 2023
Sent for Review
14 March 2023
Reviewed preprint posted
09 May 2023
Reviewed preprint revised
17 July 2023
Version of Record published
07 August 2023
Reviewing Editor:
Ying- Hui
Fu, University of California, San
Francisco, San Francisco, United
States
Copyright Barlow
et al
. This
article is distributed under the
terms of the Creative Commons
Attribution License, which
permits unrestricted use and
redistribution provided that the
original author and source are
credited.
Research article
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Barlow
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. eLife 2023;12:RP87521. DOI: https://doi.org/10.7554/eLife.87521
2 of 29
2011
). The power of screening approaches is perhaps best exemplified by the first forward genetic
sleep screen, which identified the potassium channel
shaker
as a critical sleep regulator in
Drosophila
(
Cirelli et al., 2005
). This result continues to have a lasting impact on the field as not only did subse-
quent sleep screening efforts uncover the novel Shaker regulator
sleepless
(
Koh et al., 2008
), but
investigations into Shaker’s beta subunit Hyperkinetic ultimately revealed a critical role for this redox
sensor linking metabolic function to sleep (
Bushey et al., 2007
;
Kempf et al., 2019
).
Disparate screening strategies across model organisms continue to unveil novel sleep modula-
tors in both invertebrate and vertebrate model systems. For example, the roles of RFamide receptor
DMSR-
1 in stress-
induced sleep in
C. elegans
(
Iannacone et al., 2017
) and SIK3 kinase in modu-
lating sleep homeostasis in mice (
Funato et al., 2016
) were identified in genetic screens. Moreover,
a gain-
of-
function screening strategy in
Drosophila
revealed the novel sleep and immune regulator,
nemuri
(
Toda et al., 2019
), and a zebrafish overexpression screen uncovered the secreted neuropep-
tides neuromedin U and neuropeptide Y, which decrease and increase sleep, respectively (
Chiu et al.,
2016
;
Singh et al., 2017
). The success of screening strategies in revealing novel sleep-
wake regula-
tory genes suggests that more sleep signals likely remain to be discovered.
One of the lessons from these genetic screens is that many of the uncovered genes play conserved
roles across species. For example, Shaker also regulates mammalian sleep (
Douglas et al., 2007
) and
RFamides induce sleep in worms, flies, and vertebrates (
Lee et al., 2017
;
Lenz et al., 2015
). Never
-
theless, not every invertebrate sleep-
regulatory gene has a clear vertebrate homolog, while some
human sleep/wake regulators, such as the narcolepsy-
associated neuropeptide hypocretin/orexin
(
Chemelli et al., 1999
;
Lin et al., 1999
;
Peyron et al., 2000
;
Sakurai, 2013
), lack invertebrate ortho-
logs. Therefore, genetic sleep screens in vertebrates are likely to provide added value in uncovering
additional regulatory components required to control the initiation and amount of sleep in humans.
While sleep screening in mammals is feasible (
Funato et al., 2016
), it remains an expensive and
technically challenging endeavour. With its genetic tractability, availability of high-
throughput sleep
assays (
Rihel and Schier, 2013
), and conserved sleep genetics, such as the hypocretin, melatonin,
locus coeruleus, and raphe systems (
Gandhi et al., 2015
;
Singh et al., 2015
;
Oikonomou et al.,
2019
;
Prober et al., 2006
), the larval zebrafish is an attractive vertebrate system for sleep screens.
We took advantage of a collection of zebrafish lines that harbour viral insertions in >3500 genes
(
Varshney et al., 2013
) to perform a targeted genetic screen. We identified a short-
sleeping mutant,
dreammist
, with a disrupted novel, highly conserved vertebrate gene that encodes a small single-
pass
transmembrane protein. Sequence and structural homology to the Na
+
/K
+
pump regulator FXYD1/
Phospholemman suggests that Dreammist is a neuronal-
expressed member of a class of sodium pump
modulators that is important for regulating sleep-
wake behaviour.
Results
Reverse genetic screen identifies
dreammist,
a mutant with decreased
sleep
We used the ‘Zenemark’ viral insertion-
based zebrafish gene knock-
out resource (
Varshney et al.,
2013
) to perform a reverse genetic screen to identify novel vertebrate sleep genes. This screening
strategy offers several advantages compared to traditional chemical mutagenesis-
based forward
genetic screening approaches. First, unlike chemical mutagenesis, which introduces mutations
randomly, viral insertions tend to target the 5
end of genes, typically causing genetic loss of func-
tion (
Sivasubbu et al., 2007
). Second, because the virus sequence is known, it is straightforward to
map and identify the causative gene in mutant animals. Finally, since viral insertions in the Zenemark
collection are already mapped and sequenced, animals harbouring insertions within specific gene
classes can be selected for testing (
Figure 1—figure supplement 1A
). This allowed us to prioritise
screening of genes encoding protein classes that are often linked to behaviour, such as G-
protein-
coupled receptors, neuropeptide ligands, ion channels, and transporters (
Figure 1—source data 1
).
For screening, we identified zebrafish sperm samples from the Zenemark collection (
Varshney
et al., 2013
) that harboured viral insertions in genes of interest and used these samples for in vitro
fertilisation and the establishment of F2 families, which we were able to obtain for 26 lines. For each
viral insertion line, clutches from heterozygous F2 in-
crosses were raised to 5 days post-
fertilisation
(dpf) and tracked using videography (
Figure 1—figure supplement 1A
) to quantify the number and
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duration of sleep bouts (defined in zebrafish larvae as inactivity lasting 1 minute or longer;
Prober
et al., 2006
) and waking activity (time spent moving per active bout) over 48 hr. The genotypes of
individual larvae were determined by PCR after behavioural tracking, with each larva assigned as wild
type, heterozygous, or homozygous for a given viral insertion to assess the effect of genotype on
sleep/wake behaviour. While most screened heterozygous and homozygous lines had minimal effects
on sleep-
wake behavioural parameters (
Figure 1—figure supplement 1B and C
), one homozygous
viral insertion line,
10543/10543,
had a reduction in daytime sleep (
Figure 1—figure supplement 1B
)
and an increase in daytime waking activity (
Figure 1—figure supplement 1C
) relative to their wild-
type sibling controls. We renamed this
10543
viral insertion line
dreammist
(
dmist
).
In follow-
up studies, we observed that animals homozygous for the viral insertion at this locus (
dmis-
t
vir/vir
) showed a decrease in sleep during the day and a trend to sleep less at night compared to their
wild-
type siblings (
dmist
+/+
) (
Figure 1A
).
dmist
mutants had an almost 50% reduction in the average
amount of daytime sleep (
Figure 1C
) due to a decrease in the number of sleep bouts (
Figure 1D
),
whereas the sleep bout length at night was significantly reduced (
Figure 1E
).
dmist
vir/vir
larvae also
exhibited significantly increased daytime waking activity, which is the locomotor activity while awake
(
Figure 1B and F
). Because Zenemark lines can contain more than one viral insertion (17.6% of lines
have ≥2 insertions;
Varshney et al., 2013
), we outcrossed
dmist
vir/+
fish to wild-
type fish of the AB-
TL
background and retested
dmist
mutant fish over several generations. Normalising all the behavioural
parameters to
dmist
+/+
controls with a linear mixed effects (LME) model showed consistent sleep
changes in
dmist
vir/vir
fish over five independent experiments (
Figure 1G
). The
dmist
vir/vir
larvae consis-
tently show a >50% decrease in sleep during the day due to a significant reduction in the number
and duration of sleep bouts, as well as a large increase in waking activity (
Figure 1G
). The
dmist
vir/vir
mutants also had a significant reduction in sleep at night compared to wild-
type siblings (
Figure 1G
).
These effects on sleep and wakefulness are not due to alterations in circadian rhythms as behavioural
period length in fish that were entrained and then shifted to free-
running constant dark conditions was
unaffected in
dmist
vir/vir
compared to wild-
type sibling larvae (
Figure 1—figure supplement 2A–C
).
The
dmist
gene encodes a novel, small transmembrane protein
Having identified a sleep mutant, we next sought to investigate the target gene disrupted by the viral
insertion. Line
10543
(
dmist
vir
) was initially selected for screening due to a predicted disruption of a
gene encoding a serotonin transporter (
slc6a4b
) on chromosome 5. However, mapping of the
dmist
viral insertion site by inverse-
PCR and sequencing revealed that the virus was instead inserted into
the intron of a small two-
exon gene annotated in the Zv6 genome assembly as a long intergenic non-
coding RNA (lincRNA; gene transcript ENSDART00000148146, gene name
si:dkey234h16.7
), which
lies approximately 6 kilobases (kb) downstream of the
slc6a4b
gene in zebrafish. At least part of this
region is syntenic across vertebrates, with a small two-
exon gene identified adjacent to the genes
ankrd13a
and
GIT
in several vertebrates, including human and mouse (
Figure 2A
). Amplifying both
5
and 3
ends of zebrafish
si:dkey234h16.7
and mouse E13.5
1500011B03- 001
transcripts with Rapid
Amplification of cDNA ends (RACE) confirmed the annotated zebrafish and mouse transcripts and
identified two variants with 3
untranslated regions (3
UTR) of different lengths in zebrafish (
Figure 2—
figure supplement 1B
). To test whether the viral insertion in
dmist
vir
/
vir
disrupts expression of
si:d-
key234h16.7
or neighbouring genes, we performed quantitative analysis of gene transcript levels in
wild type and mutant
dmist
larvae by RT-
qPCR. This revealed that the
dmist
viral insertion caused a
>70% reduction in the expression of
si:dkey234h16.7
while the expression of the most proximal 5
or 3
flanking genes,
slc6a4b_Dr
and
ankrd13a_Dr
, were unaffected (
Figure 2B
,
Figure 2—figure supple-
ment 1A
). Since this reduced expression is most consistent with
si:dkey234h16.7
being the causal
lesion of the
dmist
mutant sleep phenotype, we renamed this gene
dreammist
(
dmist
).
Computational predictions indicated that the
dmist
transcripts contain a small open-
reading frame
(ORF) encoding a protein of 70 amino acids (aa) (
Figure 2C
). Querying the human and vertebrate
protein databases by BLASTp using the C-
terminal protein sequence of Dmist identified orthologs
in most vertebrate clades, including other species of teleost fish, birds, amphibians, and mammals
(
Figure 2A, C
). All identified orthologues encoded predicted proteins with an N-
terminal signal
peptide sequence and a C-
terminal transmembrane domain (
Figure 2C
). The peptide sequence iden-
tity across orthologs ranged from 38 to 84%, with three peptide motifs (QNLV, CVYKP, RRR) showing
high conservation across all vertebrates and high similarity for many additional residues (
Figure 2C
,
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A)
B)
C)
G)
2
4
6
8
10
12
14
Sleep bouts (#/hr)
***
ns
p=0.06
0
0
2
4
6
8
10
Sleep bout length (min)
ns
*
12
0
2
4
6
8
10
12
14
Waking activity
(s/min)
**
ns
16
Sleep
# Sleep
bouts
Sleep
bout
length
Waking
activity
+/+
vs
vir/vir
vir/+
vs
vir/vir
***
**
***
*
***
ns
*
ns
*
ns
***
*
**
**
ns
ns
-50
-40
-30
-20
-10
0
10
20
30
% Effect Sizes relative to WT
dmist
vir/vir
, n=77
dmist
vir/+
, n=208
Sleep (min/hr
)
0
10
20
30
40
50
+/+
vir/+
vir/vir
60
**
p=0.08
+/+
vir/+
vir/vir
+/+
vir/+
vir/vir
+/+
vir/+
vir/vir
D)
E)
F)
8
7
6
5
4
3
2
1
0
14
0
14 0 14 0
night 4
day 5 night 5 day 6 night
6
Sleep (min/10 min)
Zeitgeber
Time (hr)
12
10
8
6
4
2
0
14
0
14 0 14
0
night 4
day 5 night 5 day 6 night
6
ih
t5
ht
5
ih
t5
ih
5
h
ih
h
ih
6
h
h
Wa
king ac
tivity (s/min)
Zeitgeber
Time (hr)
dmist
vir/vir
, n=19
dmist
vir/+
, n=84
dmist
+/+
, n=38
+/+
vir/+
vir/vir
+/+
vir/+
vir/vir
+/+
vir/+
vir/vir
+/+
vir/+
vir/vir
Figure 1.
A viral insertion mini-
screen identifies a short-
sleeping mutant,
dreammist
. (
A, B
) Mean ± SEM sleep (
A
) and waking activity (
B
) of progeny
from
dmist
vir/+
in-
cross from original screen. White blocks show day (lights on) and grey blocks show night (lights off). Data is combined from two
independent experiments. n indicates the number of animals. (
C–F
) Analysis of sleep/wake architecture for the data shown in (
A, B
). (
C
) Quantification
of total sleep across 2 d and nights shows decreased day and night sleep in
dmist
vir/vir
. Analysis of sleep architecture reveals fewer sleep bouts during
Figure 1 continued on next page
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Figure 2—figure supplement 1D
). Additional searches by tBLASTn failed to identify any non-
vertebrate
dmist
orthologs. In summary, we found that the
dreammist
gene, the expression of which
is disrupted in
dmist
vir/vir
fish with sleep phenotypes, encodes a protein of uncharacterised function
that is highly conserved across vertebrates at both the genomic and molecular levels.
Genetic molecular analysis of
dmist
expression in zebrafish and mouse
Because the viral insertion disrupts
dmist
throughout the animal’s lifetime, we examined both the
developmental and spatial expression of
dmist
to assess when and where its function may be required
for normal sleep. Using the full-
length transcript as a probe (
Figure 2—figure supplement 1B
), we
performed in situ hybridisation across embryonic and larval zebrafish development. Maternally depos-
ited
dmist
was detected in early embryos (two-
cell stage) prior to the maternal to zygotic transition
(
Giraldez et al., 2006
;
Figure 2D
). Consistent with maternal deposition of
dmist
transcripts, inspec-
tion of the 3
end of the
dmist
gene revealed a cytoplasmic polyadenylation element (‘TTTTTTAT’) that
is required for zygotic translation of maternal transcripts (
Villalba et al., 2011
). At 24 hpf, transcripts
were detected in regions that form the embryonic brain, such as ventral telencephalon, diencephalon,
and cerebellum, and in the developing eye (
Figure 2D
). By 5 dpf,
dmist
transcripts were detected
throughout the brain (
Figure 2D
). To test whether
dmist
transcripts are under circadian regulation, we
performed RT-
qPCR in fish that were entrained and then shifted to free-
running constant dark condi-
tions. In contrast with the robust 24 hr rhythmic transcription of the circadian clock gene
per1
, we did
not detect any changes in
dmist
expression throughout the 24 hr circadian cycle (
Figure 1—figure
supplement 2D
).
Consistent with brain expression in larval zebrafish, we identified the expression of
Dmist_Mm
in
a published RNAseq dataset of six isolated cell types from mouse cortex (
Zhang et al., 2014
). We
confirmed that
Dmist_Mm
is specifically enriched in neurons by hierarchical clustering of all 16,991
expressed transcripts across all six cells types, which demonstrated that
Dmist_Mm
co-
clusters with
neuronal genes (
Figure 2—figure supplement 1E
). Pearson correlation of
Dmist_Mm
with canon-
ical markers for the six cell types showed that
Dmist_Mm
expression is highly correlated with other
neuronal genes but not genes associated with microglia, oligodendrocytes, or endothelia. This result
indicates that
dmist
is specifically expressed in neurons in both zebrafish and mouse (
Figure 2—figure
supplement 1F
).
Dmist localises to the plasma membrane
Although the
dmist
gene encodes a conserved ORF with a predicted signal peptide sequence and
transmembrane domain (
Figure 2C
,
Figure 2—figure supplement 1G–I
), we wanted to confirm this
small peptide can localise to the membrane, and if so, on which cellular compartments. To test these
computational predictions, we transiently co-
expressed GFP-
tagged Dmist (C-
terminal fusion) with a
marker for the plasma membrane (myr-
Cherry) in zebrafish embryos. Imaging at 90% epiboly revealed
Dmist-
GFP localised to the plasma membrane (
Figure 2E
). Conversely, introducing a point mutation
into Dmist’s signal peptide cleavage site (DmistA22W-
GFP) prevented Dmist from trafficking to the
plasma membrane, with likely retention in the endoplasmic reticulum (
Figure 2F
). Together, these
the day (
D
) and shorter sleep bouts at night (
E
) in
dmist
vir/vir
compared with sibling controls. (
F
) Daytime waking activity is also increased in
dmist
vir/vir
. The
black lines show the mean ± SEM, except in (
E
), which labels the median ± SEM. *p<0.05, **p<0.01, ***p<0.001; ns p>0.05; one-
way ANOVA, Tukey’s
post hoc test. (
G
) Combining five independent experiments using a linear mixed effects model with genotype as a fixed effect and experiment as a
random effect reveals
dmist
vir/vir
larvae have decreased total sleep and changes to sleep architecture during both the day and night compared to
dmist
+/+
siblings. Plotted are the genotype effect sizes (95% confidence interval) for each parameter relative to wild type. Shading indicates day (white) and night
(grey). p-
Values are assigned by an
F
-
test on the fixed effects coefficients from the linear mixed effects model. *p<0.05, **p<0.01, ***p<0.001, ns p>0.05.
n indicates the number of animals.
The online version of this article includes the following source data and figure supplement(s) for figure 1:
Figure supplement 1.
A viral insertion screen for sleep-
wake regulators.
Figure supplement 2.
dmist
vir/vir
fish are hyperactive and have normal circadian rhythms.
Source data 1.
Gene selection for screening.
Figure 1 continued
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Predicted T
ransmembrane
Predicted Signal Peptide
Danio rerio
Takifugu rupribes
Tetraodon nigroviridis
Xenopus laevis
Gallus gallus
Mus musculus
Homo sapiens
Danio reri
o
Chr5
slc6a4b
ankrd13a
Mus musculu
s
Chr5
Homo sapiens
Chr12
git2a
viral insert
Ankrd13
GIT
IFT8
RIKEN cDNA
1500011
B03-001
ANKRD13
IFT8
GIT
c12orf76-003
si:dkey-234h16.
7
‘dreammist
8.8kbp
6.2kbp
Te l
Dien
dmist
lateral
dorsal
5dpf
24hp
f
2 cell
Dien
R
1
-
6
Te l
eye
R1-6
AP
dorsal
+/+
vir/+
vir/vir
0.2
0.6
1
1.4
Relativ
e
expression (a.u.)
dmist
, n=3
*
**
+/+
vir/+
vir/vir
0.2
0.6
1
1.4
1.8
ankrd13a
, n=2
Relative
expression (a.u.)
ns
+/+
vir/+
vir/vir
0.2
0.6
1
1.4
1.8
slc6a4b
, n=2
Relativ
e
expression (a.u.)
ns
A)
B)
C)
D)
E)
F)
myr-Cherry
CMV
:dmistA22W
-GFP
myr-Cherry;
CMV:
dmistA22W
-GFP
myr-Cherry
CMV
:dmist-GFP
myr-Cherry;
CMV
:dmist-GFP
Figure 2.
dmist
encodes a conserved vertebrate single-
pass transmembrane protein. (
A
)
dmist
mutants harbour a viral insertion in the first intron of
si:key- 234h16.7. dmist
is syntenic with
Ankrd13
and
GIT
orthologs in mouse, human, and zebrafish. (
B
) RT- qPCR of
dmist
(red) show reduced expression
of
dmist
and not the 5
and 3
flanking zebrafish genes,
slc6a4b
(cyan) and
ankrd13a
(blue), in
dmist
vir/vir
larvae compared to
dmist
vir/+
and
dmist
+/+
siblings. **p<0.01, *p<0.05; ns p>0.05; one-
way ANOVA, Tukey’s post hoc test. Data shows mean ± SEM normalised to the wild-
type mean. (
C
)
dmist_Dr
Figure 2 continued on next page