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Phylogenetic Analysis and Expression of Zebrafish Transient Receptor Potential Melastatin Family Genes

Authors, affiliations and grants: Edda Kastenhuber1, Matthias Gesemann1, Michaela Mickoleit1,2, Stephan C. F.

Neuhauss1*

1Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190,

8057 Zurich, Switzerland

2Present address: Max-Planck-Institute of Molecular Cell Biology and Genetics,

Pfotenhauerstrasse 108, 01307 Dresden, Germany

*Correspondence to: Stephan C. F. Neuhauss, Institute of Molecular Life Sciences,

University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. E-mail:

stephan.neuhauss@imls.uzh.ch Telephone: +41 (0)44 63 56040 Fax: +41 (0)44 635

6817

Grant sponsor: EMBO ALTF 326-2010, Swiss National Science Foundation

31003A_135598, EU FP7 RETICIRC

Running title: Zebrafish TRPM family

Key words: Danio rerio; TRPM ion channels; sensory neurons; pronephros

Key findings: The zebrafish TRPM family consists of 11 genes.

Zebrafish trpm genes show dynamic expression pattern over embryonic and

larval stages observed.

Expression was identified in both excitable and non-excitable cells, reflecting

the broad functional range of TRP channels.

Zebrafish trpm expression was found in cell clusters involved in sensory

information processing, ion homeostasis, and osmolarity as well as in the

developing brain.

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Abstract: Background: The transient receptor potential melastatin (TRPM) gene family belongs to

the superfamily of non-selective TRP ion channels. TRP channels are cellular sensors,

detecting a multitude of inputs, including temperature, light and chemical and

mechanical stimuli. Recent studies revealed diverse roles during development, linking

TRP channels to differentiation, proliferation, cell motility, cell death and survival. A

detailed description of this gene family in the zebrafish is still missing.

Results: Phylogenetic analysis revealed 11 trpm genes in the zebrafish genome. The

zebrafish orthologs of mammalian TRPM1 and TRPM4 are duplicated and

quadruplicated, respectively, and TRPM8, a cold sensitive channel has been lost in

zebrafish. Whole-mount in situ hybridization experiments revealed dynamic expression

patterns of trpm genes in the developing embryo and early larva. Transcripts were

mainly found in neural cell clusters, but also in tissues involved in ion homeostasis.

Conclusions: Our results suggest a role of TRPM channels in sensory information

processing, including vision, olfaction, taste, and mechanosensation. An involvement in

developmental processes is likely, as some trpm genes were found to be expressed in

differentiating cells. Our data now provides a basis for functional analyses of this gene

family of ion channels in the vertebrate model organism Danio rerio.

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Introduction: The transient receptor potential (TRP) channel was first described in the fruit fly

Drosophila melanogaster after the identification of a mutant fly that showed a transient

instead of a sustained response to continuous light exposure (Montell and Rubin, 1989;

Hardie and Minke, 1992). Subsequent phylogenetic analysis led to the discovery of a

whole superfamily of ion channels that are conserved throughout the animal kingdom.

The human TRP family consists of 27 genes clustering into six subfamilies (TRPC,

TRPM, TRPV, TRPA, TRPP, and TRPL). Although single channels can differ strongly

in their physiological characteristics, they all share a common structure consisting of six

transmembrane domains, the pore forming region between S5 and S6 and intracellular

amino- and carboxy-termini. Functional channels are known to form homo- or

heterotetramers, displaying a large diversity of ion selectivity, activation mode, and

gating mechanisms (reviewed for example by Clapham et al., 2001; Montell, 2005;

Venkatachalam and Montell, 2007; Nilius and Owsianik, 2011).

Expression of individual members was found in most tissues, underscoring their

perceived role as multifunctional cellular sensor proteins. Indeed, functional analyses of

TRP ion channels revealed their participation in all kinds of sensory perception:

thermo-, mechano-, and chemosensation, as well as nociception and light perception

(summarized in Voets et al., 2005 and Damann et al., 2008). Another central role of

TRP channels is the maintenance of ion homeostasis and osmoregulation. Therefore

they have been associated with diverse medical conditions, among them polycystic

kidney disease, hypomagnesemia with secondary hypocalcemia (HSH), idiopathic

hypercalciuria, cardiac hypertrophy, and hypertension as just a few examples

(Woudenberg-Vrenken et al., 2009; Dietrich and Gudermann, 2011). Additionally, many

TRP channels are involved in diverse developmental processes such as cell death,

proliferation, axon outgrowth and guidance, as well as synaptic plasticity (Dadon and

Minke, 2010; Santoni et al., 2011; Vennekens et al., 2012).

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The subfamily of TRP melastatin (TRPM) channels shows structural and functional

diversity to a similar extent as the whole superfamily. It comprises eight members in

mammals: TRPM1 to TRPM8. Of these TRPM2, TRPM6 and TRPM7 are unique

amongst the TRP channels in that they possess an enzymatically active protein domain

in their intracellular structure (Perraud et al., 2001; Runnels, 2011). TRPM4 and

TRPM5 channels do not show any permeability for Ca2+ in contrast to the other TRP

channels, although their activation is highly dependent on increases in intracellular

Ca2+ levels (reviewed by Guinamard et al., 2011). Currently little information is

available for the zebrafish TRPM family.

The teleost Danio rerio is one of the best established genetic and behavioral vertebrate

model organisms. In zebrafish TRP channel function in vivo can be studied using a

variety of tools, including morpholino knockdown, zinc finger nuclease, TALEN

(transcription activator-like effector nuclease), TILLING (targeted induced local lesion in

genomes) and transgenesis technologies (reviewed in Huang et al., 2012; Sugano and

Neuhauss, 2013). In this study, we identified and cloned cDNAs for 11 trpm genes and

studied their expression over development. We were able to identify excitable and non-

excitable tissues expressing trpm channels. Our findings suggest a variety of functional

implications for zebrafish TRPM channels and form the basis for further investigations.

Results and Discussion: The zebrafish trpm family:

We used the verified Homo sapiens and Mus musculus sequences of TRPM channels

to search the ensemble databases in order to identify trpm genes of Danio rerio. While

mouse and human genomes contain eight TRPM members, we found 11 zebrafish

trpm genes (figure 1). We ascertained a single zebrafish ortholog for the following

mammalian TRPM channels: TRPM2, TRPM3, TRPM5, TRPM6, and TRPM7. Two

zebrafish trpm genes, namely trpm1 and trpm4, were found to have multiple paralogs.

The two trpm1 paralogs, trpm1a and trpm1b, were located to different chromosomes

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and hence are likely to be the result of the teleost specific R3 whole genome

duplication. In addition to the three zebrafish trpm4 paralogs described in Saito and

Shingai, 2006, we identified a fourth trpm4 gene. Whereas trpm4a was found on

chromosome 3, the three other paralogs, trpm4b1, trpm4b2, and trpm4b3, are situated

on chromosome 12. Phylogenetic and synteny analyses indicate that trpm4a and one

of the trpm4bs originate from the R3 genome duplication; whereas the other two

paralogs seem to be the result of tandem gene duplications (figure 1A, C). Based on

their high sequence similarity, these tandem duplications likely happened very recently.

In agreement with Saito and Shingai, 2006, we failed to identify a zebrafish trpm8

ortholog, supporting their conclusion that trpm8 is indeed absent from the zebrafish

genome. In mammalian species TRPM8 has been described as a menthol and cold

sensitive TRP channel involved in body and ambient temperature perception below

25°C (McKemy et al., 2002; Peier et al., 2002).

Using sequence information from our in silico analysis, we amplified and

subsequently cloned the open reading frames for the zebrafish trpm genes. Using

these cDNA templates, we performed whole mount in situ hybridization (WISH) to

analyze the expression pattern of trpm genes in the developing zebrafish embryo

ranging from 24 hours to 5 days post fertilization. With the exception of trpm6 we found

distinct expression patterns for trpm genes in the developing zebrafish. However, as

human TRPM6 expression was exclusively found in kidney and intestine (Schlingmann

et al., 2002; Walder et al., 2002), we assume that the expression level in these organs

at the stages analyzed is too low to be visualized. An overview of the zebrafish trpm

family expression pattern observed throughout the developmental stages analyzed is

given in table 1.

Expression of trpm1 genes:

The founding member of the TRPM family, TRPM1 (melastatin), was initially identified

in melanoma cell lines and its expression levels seem to be inversely correlated to

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metastasis of these tumors (Duncan et al., 1998). Here we describe the expression

pattern of the two zebrafish paralogs, trpm1a and trpm1b. We observed trpm1a

expression predominately in melanogenic cells of the neural crest (NC) at 24 hpf (see

figure 2 A-C). These cells are most likely future melanophores, the zebrafish

equivalents to mammalian melanocytes, as they are dispersed over the dorsal tail and

all over the trunk and head of the embryo. The expression of trpm1a in melanophores

is sustained at 48 hpf (figure 2 D, E), and was also found in single cells mainly on the

ventral side of 3 and 5 dpf larvae (figure 2 H). It has been shown that TRPM1 regulates

positively Ca2+ uptake in human epidermal cells, thereby influencing melanocyte

development and melanin accumulation (Devi et al., 2009). It might well be that the

zebrafish ortholog TRPM1a plays a similar role in melanophore growth and

differentiation. Additionally to the expression in epidermal pigment cells, trpm1a was

found to be expressed in different cell types of the retina. In zebrafish embryos at 24

and 48 hpf, numerous cells all over the eye and especially around the lens were

labeled by WISH (figure 2 F). The retinal region around the lens is called the ciliary

marginal zone (CMZ) and contains retinal stem cells. Expression in the ciliary margin is

maintained at least until 5 dpf (figure 2 G, H, and K). Expression of a TRPM1 channel

in retinal progenitors has not been described for any other species so far. Recent

studies on TRPC1, another TRP channel from the canonical family, showed its

involvement in proliferation of embryonic and adult neuronal stem cells, as well as

oligodendrocyte precursors. Its function as a store-operated Ca2+ channel is thought to

be crucial for maintaining intracellular Ca2+ levels required for proliferation (Fiorio Pla et

al., 2005; Paez et al., 2011; Li et al., 2012). Moreover, a role of TRPC5 in the transition

from proliferation to differentiation has been revealed in cultures of neural progenitors

(Shin et al., 2010). Although an involvement of TRPM1 channels in neural stem cell

development has not been reported before, it is conceivable that zebrafish TRPM1a

has a functional role in retinal development. The trpm1a expressing cells in the outer

retinal neuroepithelium of embryonic stages most likely represent future retinal pigment

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epithelial (RPE) cells (figure 2 B-D). At 3 dpf, expression of trpm1a started in the inner

nuclear layer (INL) of the retina (figure 2 G), which contains bipolar cells amongst other

cell types. Larvae at 5 dpf still expressed the gene in the INL, as well as in the RPE

(figure 2 J, K).

Compared to trpm1a, the second zebrafish paralog, trpm1b, shows over development

a more restricted and less dynamic expression pattern. trpm1b is initially also

expressed in a subset of cells in the outer retinal neuroepithelium (figure 2 L-N),

presumably labeling RPE progenitor cells. A small band of trpm1b expressing cells is

additionally observed in the ventral eye transiently at 48 hpf (figure 2 M). These cells

are located in the choroid fissure that has not yet been closed at this stage. Similar to

trpm1a, expression of trpm1b starts in cells of the INL at 3 dpf and is also found in

differentiated RPE cells (figure 2 O-R). Retinal expression of both trpm1 paralogs

correlates well with TRPM1 expression in the human eye (Klooster et al., 2011).

Recently, TRPM1 became known for its function in the light perceiving signaling

pathway of the retina. It was found to be the long missing ion channel gated by the

metabotropic glutamate receptor mGluR6 in ON bipolar cells (Morgans et al., 2009;

Shen et al., 2009; Koike et al., 2010). A considerable proportion of human patients

suffering from complete congenital stationary night blindness show mutations in the

TRPM1 channel gene (Audo et al., 2009; Li et al., 2009; van Genderen et al., 2009).

Expression of trpm2:

Mammalian TRPM2 expression has been studied mainly by RT-PCR experiments.

Transcripts were found in a variety of tissues, such as brain, immune cells, vascular

endothelial cells, pancreas and other inner organs (summarized in Sumoza-Toledo and

Penner, 2011). WISH of zebrafish trpm2 showed a dynamic expression pattern over

the course of development. At 24 hpf the gene is expressed in lens tissue and

motoneurons (MN, figure 3A-C). Over time lens expression becomes more restricted

leaving expression only in a specific spot between the lens and RGC layer, close to the

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point where the optic nerve exits the retina (optic nerve head (ONH), figure 3 E, G, K).

Motoneurons lose trpm2 expression already after 24 hpf. 48 hpf onwards expression

appears in a subset of olfactory sensory neurons (figure 3 D, H, K, N, P). We also

detected trpm2 transcripts in bilateral clusters in the hindbrain Past day 3 this

expression becomes rather diffuse and diminishes more and more. At 48hpf trpm2

expression was also observed in cells of the trigeminal and the posterior lateral line

ganglia (figure 3 F-H, L, N, O). This expression extends to all cranial sensory ganglia

by day 3 and 5 (figure 3 L, N, O). Little is known about the function of TRPM2 during

development. Therefore, it is striking that we found trpm2 transcripts primarily in

sensory placodes of embryonic and larval zebrafish. The function of TRPM2 in the

developing embryo is an interesting subject for future studies. As TRPM2 belongs to

the thermosensitive TRP channels and is best known for its involvement in oxidative

stress response and consequent cell death (Hara et al., 2002; Zhang, 2003; Takahashi

et al., 2011) as well as in synaptic plasticity of hippocampal neurons (Olah et al., 2009;

Xie et al., 2011) it will be interesting to analyze TRPM2 functions in the described

zebrafish expression domains.

Expression of trpm3:

Expression of human TRPM3 is known to be highly enriched in kidney compared to

lower expression in brain, spinal cord and testis (Grimm et al., 2003; Lee et al., 2003).

Surprisingly, RT-PCR and Northern blot analyses in mouse showed high expression in

various brain areas, such as the telencephalon, cerebellum, basal ganglia, choroid

plexus and hippocampus but comparatively low expression in kidney and other organs.

However, distinct mouse strains showed significant differences in their expression

levels of TRPM3 in the telencephalon (Oberwinkler et al., 2005; Kunert-Keil et al.,

2006). The zebrafish ortholog of the TRPM3 gene is found to be expressed by neural

crest cells in the head and dorsal tail region of embryos 24 hpf (figure 4 A, C).

Expression is also present in the habenula (Ha; figure 4 B) and lens tissue (L; figure 4

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C). Whereas NC cell expression ceases after 24 hpf, expression in the lens and

habenula is retained over all stages observed. From 48 hpf onwards, more and more

neurons of the central nervous system (CNS) start to express this gene. We observed

expression in the epiphysis (E) and dorsal thalamus (DT), together with a rather broad

expression in the hindbrain (Hb; figure 4D-E) at 48 hpf (figure 4 D, E). Moreover, lens

expression of trpm3 becomes restricted to the lens epithelial (LEp) layer (figure 4 F).

CNS expression gets more pronounced over development. While expression in the

epiphysis seems to be lost after 3 dpf, we found additional trpm3 staining in the optic

tectum (TeO) and choroid plexus (CP, figure 4 G-L). Finally, larvae show expression in

a subset of ganglion cells of the retina at 5 dpf (GCL, figure 4 M). It is noteworthy that

the expression pattern of trpm3 partially overlaps with the expression pattern of

mglur6a, which was found in retinal ganglion cell layer, habenula, optic tectum and

thalamus as well (Haug et al., 2013). This suggests the possibility that a similar

glutamate signaling pathway as described for ON bipolar cells may be active in these

neurons as well. Moreover, a recent study showed the localization of TRPM3 in

Purkinje cells of the neonatal rat cerebellar cortex and its capability to enhance

glutamatergic transmission (Zamudio-Bulcock et al., 2011). These findings suggest a

role of TRPM3 in synapse refinement during development.

Expression of trpm4 genes:

TRPM4 is one of the two TRP channels that is selective for monovalent cations and

activated by high intracellular Ca2+ levels. In humans the channel is predominantly

expressed in skeletal muscles and inner organs, including heart, kidney, liver and

pancreas (Launay et al., 2002). A similar distribution of trpm4 mRNA was observed in

mice (Kunert-Keil et al., 2006). The zebrafish ortholog trpm4a was found to be

expressed only in three distinct domains. WISH analysis of trpm4a revealed expression

to be restricted to the cloaca of the pronephros in embryos 24 hpf (figure 5 A, B). By

48hpf, the pharyngeal ectoderm (PEc) starts to express trpm4a, but this expression is

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lost again at larval stages analyzed. However, cloacal expression is sustained until at

least 5 dpf (figure 5 D, F, H), with expression even expanding into the caudal part of

the pronephros (figure 5 H). Additionally, we located trpm4a transcripts in the intestinal

bulb from 3 dpf onwards (figure 5 E, G).

This comparatively restricted expression is in contrast to the other three trpm4

paralogs, which show a broader expression pattern. As every probe for trpm4bs gave

identical results it seems likely that our probes used for the trpm4b variants are unable

to discriminate between the 3 different transcripts. Unfortunately probes against 5’

untranslated regions of trpm4bs that displayed less sequence conservation proved

unable to detect expression, probably due to their rather short length (see table 2).

Therefore, we can not determine whether transcripts of a particular trpm4b gene is

present only in a subset of the domains stained with the long trpm4b probes or is not

expressed at all. Initial expression at 24 hpf is visible in the olfactory, pronephric and

lateral line systems. More specifically, expression is detectable in olfactory sensory

neurons (figure 5 J) and the pronephric cloaca (figure 5 M). At 24hpf weak expression

is also seen in the lateral line primordium (LLP), and stronger expression can be

detected in the newly formed neuromast cells (LLN; figure 5 K, L). Although expression

in olfactory sensory neurons and neuromasts seems to decrease after 3 dpf, we could

observe WISH staining in these cells at all stages analyzed (figure 5 N-V). At

embryonic stage 48 hpf, transcripts for trpm4bs can be seen in cells of the pharyngeal

ectoderm (figure 5 O, P) and are maintained at least until 5 dpf. The gill ectoderm

(GEc) also shows expression as soon as gill development started (figure 5 R, V). Last

but not least, we observed yet another expression domain in the intestinal bulb of 5 dpf

larvae (IB; figure 5 W), comparable to the expression of trpm4a at this stage.

Expression of trpm4s in cloaca, intestine, pharynx and gill ectoderm points to a role in

ion homeostasis or osmoregulation. Moreover, expression of TRPM4 in the murine

accessory olfactory system (AOS) has been described recently and seems to be at

least partially responsible for the sustained response of neurons (Shpak et al., 2012).

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The mammalian and reptile accessory olfactory system is considered to be involved in

the fluid based chemosensation in contrast to the main olfactory system, which detects

volatile substances. Therefore it is reasonable to expect a conserved TRP expression

pattern of teleost olfactory neurons and mammalian AOS neurons. In contrast,

TRPM4b channels in the lateral line system could account for the transduction of

mechanical stimuli as specialized hair cells in the lateral line neuromasts are able to

detect water movements and vibrations comparable to hair cells in the mammalian

inner ear.

Expression of trpm5:

The expression of zebrafish trpm5 in juvenile zebrafish aged approximately 2 months

has already been described by Yoshida et al., 2007. In this study they demonstrated

expression in taste bud cells located on the lips, barbels, and headskin as well as in the

mouth cavity, gill rakers, and pharynx. In contrast to these experiments, we analyzed

trpm5 expression in the whole animal at earlier developmental stages. However, no

transcripts of trpm5 were detected at 24 and 48 hpf, respectively. In accordance with

Yoshida et al., 2007, we could confirm expression of trpm5 in taste bud cells of the

pharynx and gills to be present already at larval stages 3 and 5 dpf (figure 6 A-F) but

not in any other tissue. Its expression in taste receptor cells as well as its function in

the transduction of different taste stimuli is well established. TRPM5 channels are

activated upon ligand binding to G-protein coupled receptors of the taste receptor T1R

and T2R families and mediate perception of sweet, umami and bitter taste (Pérez et al.,

2002; Zhang et al., 2003; Damak et al., 2006). Their additional thermosensitivity is

proposed to modulate taste reception dependent on temperature (Talavera et al.,

2005). Our expression analysis together with the data of Yoshida et al., 2007 suggests

a conservation of TRPM5 in the transduction of taste stimuli among vertebrate species.

Expression of trpm7:

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TRPM7 is the best studied member of the zebrafish TRPM family so far. Its expression

has been described as rather ubiquitous with stronger expression in lens sensory

placodes, ionocytes, epiphysis, pharynx, pronephros, and Rohon-Beard neurons

between 24 – 55 hpf (Thisse, 2001; Hsiao et al., 2007; Low et al., 2011). Our

observations are in line with these data (figure 7 A-F), although expression in the spinal

cord is very weak. We extended the expression analysis to larval stages (figure 7 G-M).

TRPM7 is a well established marker for the pronephric proximal straight tubule in

zebrafish embryos (Wingert et al., 2007), however at 5 dpf larvae shows an extension

of the expression into the proximal convoluted tubule (figure 7 K-L). Additionally, we

observed trpm7 transcripts above the heart (arrowhead in figure 7 E) and in two

bilateral spots caudal to the pharynx (arrowheads in figure 7 G-H). Some zebrafish

lines carrying different mutations of the gene have been isolated in genetic screens for

pigmentation, pancreas development or locomotion deficiencies (Granato et al., 1996;

Kelsh et al., 1996; Arduini and Henion, 2004; Low et al., 2011; Yee et al., 2011). Loss

of TRPM7 usually leads to lethality between 14 and 16 dpf. Nevertheless, one semi-

viable allele was identified enabling phenotypic description into adulthood. These fish

showed kidney stone formation accompanied by severe dwarfism due to massive

disruption of bone ossification, indicative of the important role of TRPM7 in calcium and

magnesium homeostasis (Cornell et al., 2004; Elizondo et al., 2010). Larval trpm7

mutants show hypopigmentation due to a failure in melanoblast development (Arduini

and Henion, 2004; Cornell et al., 2004; McNeill et al., 2007). Moreover, trpm7 mutants

become unresponsive in escape response tests assessing mobility at 39 hpf but

recover completely after 72 hpf. The transgenic expression of trpm7 in

mechanosensitive Rohon-Beard cells of mutant larvae could rescue this behavioral

phenotype, but the transiently required role of the cation channel in the sensory

neurons is not yet clarified (Low et al., 2011). The expression pattern of TRPM7

indicates the pivotal role of the channel in ion homeostasis, and it seems possible that

all observed phenotypes of trpm7 mutants rely on this function.

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Taken together our phylogenetic and expression analyses of the TRPM family of ion

channels in zebrafish open the way for further studies on their function. As expression

was found in diverse organs and tissues, including excitable and non-excitable cells,

we expect TRPM channels to be implicated in a multitude of processes. We

concentrated on expression during embryonic and larval stages and found transcripts

in variegated cell clusters of the central and peripheral nervous system. It will be

interesting to investigate trpm genes expressed in neural cells during development and

elucidate their role in neural differentiation and proliferation, axon guidance, or synaptic

plasticity. Another interesting aspect is expression of TRPM channels in cells involved

in metabolism and homeostasis, such as the kidney, intestine, and gills. A better

understanding of their physiological functions may provide further insight into disease

mechanisms and support the development of therapies. The zebrafish offers a broad

variety of genetic and behavioral tools to assess the function of TRPM channels in vivo.

Experimental Procedures: Fish maintenance:

Zebrafish breeding and maintenance were carried out under standard conditions (26-

28°C, 14h light/10h dark) according to Westerfield, 2007. The wildtype WIK line was

used for all experiments described here. Pigmentation of embryos was avoided by

incubating them in 0.2 mM 1-phenyl-2 thiourea (Sigma).

Annotation of trpm cDNAs

As automated gene predictions within GenBank are subjected to putative annotation

errors, trpm cDNA sequences used in this study were annotated manually. Annotations

were done based on combined information obtained from expressed sequence tags

and genome databases (GeneBank, http://www.ncbi.nlm.nih.gov; Ensembl,

http://www.ensembl.org/index.html). Human and mouse sequences were used as initial

query (for more details on sequence annotation see Gesemann et al., 2010).

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Phylogenetic and synteny analyses:

The phylogenetic analysis was performed on the Phylogeny.fr platform

(http://www.phylogeny.fr/) as described previously (Gesemann et al., 2010; Haug et al.,

2013). In brief, sequences alignment was done using MUSCLE. Input sequence length

varied between 1104 and 2028 amino acids. Ambiguous regions were removed using

Gblocks and 438 conserved amino acids were used to reconstruct a maximum

likelihood tree. Branch liability was assessed using the aLRT test and svg tree file were

imported into CoralDraw (version X5; Coral Corporation Ottawa, Canada) for final

editing.

Cloning and sequencing of trpm genes:

Total RNA was extracted from a pool of 5 dpf wild-type zebrafish embryos using the

Macherey-Nagel NucleoSpin® RNA II kit. cDNA was subsequently generated

according to the manual of the Invitrogen SuperScript™ II RT-PCR kit. Fragments of

the respective trpm genes were amplified by means of polymerase chain reaction using

the oligonucleotides listed in table 2, cloned into the pCR®II-TOPO vector following the

TOPO TA Cloning® instruction manual and eventually sequenced. If necessary and

possible 5’ rapid amplification of cDNA ends was performed using the Invitrogen 5’

RACE System Version 2.0 in order to obtain cDNA fragments comprising the start

codon.

Probe synthesis and whole mount in situ hybridization:

Preparation of RNA probes and expression analyses with alkaline phosphatase-based

color reaction was performed as described previously (Hauptmann and Gerster, 2000)

with the following adaptations. The RNA probe was recovered by lithium chloride

precipitation. The duration of Proteinase K treatment was 60 min and 90 min for

embryos fixed 3 and 5 dpf, respectively. Hybridization was performed overnight at

65°C. The anti-digoxigenin antibody (Roche #11093274910) was diluted at 1:5000 with

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1% Roche blocking reagent (Roche #11096176001) in TNT buffer (100mM Tris-HCL

pH7.5, 150 mM NaCl, 0.5% Tween20). Washing steps after antibody incubation were

also done with TNT buffer. 2 mM levamisole hydrochloride (Sigma L9756) was added

to the staining buffer in order to prevent unspecific staining by endogenous alkaline

phosphatase.

Imaging:

Embryos were embedded in glycerol. Images were captured using a ColorView IIIu

camera and an Olympus BX61 widefield microscope. Levels of pictures were adjusted

and figures were assembled using Adobe Photoshop software.

Acknowledgments: We thank Kara Dannenhauer for excellent technical assistance and zebrafish care, as

well as Andreas Stäuble for help in cloning trpm4 genes.

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Figure 1:

Origin, conservation and phylogenetic relation of zebrafish TRPM channels. (A) Phylogenetic relations between TRPM channels from human mouse and zebrafish. Zebrafish (dr) TRPMs are shown in red, human (hs) TRPMs are depicted in dark gray and mouse (mm) TRPMs are given in light gray. The tree was rooted using the zebrafish TRPN1 channel as an outgroup. (B) Identity and conservation within the trpm4 subgroup. The four TRPM4 sequences zebrafish were compared with each other and with the corresponding orthologous of mouse and human. The first line represents the amino acid comparison (bold = identical, in parentheses = conserved), whereas the second line gives identities on nucleotide level. While, zebrafish TRPM4 resulting from tandem duplication events are highlighted in dark red, homologies between human and mouse TRPM4s are pointed out in orange. (C) Synteny of the zebrafish trpm4 genes. Genes flanking the trpm4 genes on the zebrafish chromosomes 3 and 12 are shown in the central part of the figure. Zebrafish chromosomes 3 and 12, harboring the trpm4 genes, have been previously reported to derive from a common protochromosome (Woods et al., 2005). Note that the genes depicted here have their counterparts on human chromosomes 10 (q23.33), 16 (p11.2, p12.3 and p13.3) 17 and 19 (q13.42) and

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that several genes found in close proximity to the human trpm4 gene (e.g. PRR12 or BRSK1) have corresponding representatives on both zebrafish chromosomes. The additional zebrafish trpm4 genes on chromosomes 12 most likely originated by tandem duplication and partial chromosome inversions of the original trpm4 duplicate leading to two additional trpm4 genes in zebrafish.

Figure 2: Expression of trpm1 channel genes, trpm1a and trpm1b, shown in whole-mount during zebrafish development. A-K: trpm1a expression with A-C: lateral (A) and dorsal (B-C) views of embryos 24 hpf; D-F: dorsal (D-E) and lateral (F) views of embryos staged 48 hpf; G-H: 3 dpf larvae shown from the ventral side; J-K: ventral (J) and lateral (K) views of larvae staged 5 dpf. L-R: trpm1b expression with L: dorsal view of embryos 24 hpf; M-N: embryos staged 48 hpf shown from the dorsal side; O-P: ventral (O) and lateral (P) views of larvae 3 dpf; Q-R: ventral (Q) and lateral (R) views of larvae 5dpf. Zebrafish are orientated with anterior to the left in all pictures but G, J, O, and Q, where anterior is to the top. Scale bars of 100 µm are indicated in the first picture of a series until changed. Abbreviations: NC = neural crest; Mel = melanoblast / -cyte; CMZ = ciliary marginal zone; INL = inner nuclear layer; RPE = retinal pigment epithelium.

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Figure 3: Dynamic expression pattern of trpm2 during zebrafish development, shown in whole-mount views. A-C: dorsal (A-B) and lateral (C) views of embryos 24 hpf; D-H: ventral (D-F) and lateral (G-H) views of embryos staged 48 hpf; J-M: 3 dpf larvae shown from the dorsal (J), ventral (K) and lateral (L-M) side, respectively; N-P: ventral (N), lateral (O), and frontal (P) views of larvae staged 5 dpf. Orientation of zebrafish is with anterior to the left in all pictures but P showing a frontal view. Scale bars of 100 µm are indicated in the first picture of a series until changed. Abbreviations: L = lens; Mn = motoneurons; OSN = olfactory sensory neurons; TG = trigeminal ganglia; PLG = posterior lateral line ganglia; Hb = hindbrain; ONH = optic nerve head; CSG = cranial sensory ganglia.

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Figure 4: Expression of trpm3 in whole-mount zebrafish during embryonic and larval stages. A-C: lateral (A-B) and dorsal (C) views of embryos 24 hpf; D-F: lateral (D, F) and dorsal (E) views of embryos staged 48 hpf; G-J: 3 dpf larvae shown from the dorsal (G-H) and lateral (J) side, respectively; K-M: lateral (K), dorsal (L), and ventral (M) views of larvae staged 5 dpf. Zebrafish are orientated with anterior to the left in all pictures but M, where anterior is to the top. Scale bars of 100 µm are indicated in the first picture of a series until changed. Abbreviations: NC = neural crest; Ha = habenula; E = epiphysis; DT = dorsal thalamus; Nhp = neurohypophysis; Hb = hindbrain; LEp = lens epithelium; TeO = optic tectum; CP = choroid plexus; GCL = ganglion cell layer.

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Figure 5: Expression of trpm4 channel genes, trpm4a and trpm4b, shown in whole-mount during zebrafish development. Expression patterns of both, trpm4c and trpm4d, are identical to trpm4b. A-K: trpm4a expression with A-B: lateral (A) and ventral (B) views of embryos 24 hpf; C-D: lateral views of embryos staged 48 hpf; E-F: 3 dpf larvae shown laterally; G-H: lateral views of larvae staged 5 dpf. J-W: trpm4b expression with J-M: dorsal (J-K) and lateral (L-M) views of embryos 24 hpf; N-P: embryos staged 48 hpf shown from the lateral (N, P) and ventral (O) side; Q-T: lateral (Q, S-T) and ventral (R) views of larvae 3 dpf; U-W: lateral (U) and ventral (V-W)) views of larvae 5dpf. Zebrafish are orientated with anterior to the left. Scale bars of 100 µm are indicated in the first picture of a series until changed. Abbreviations: Cl = cloaca; PEc = pharynx ectoderm; IB = intestinal bulb; OSN = olfactory sensory neurons; LLN = lateral line neuromast; LLP = lateral line primordium; GEc = gill ectoderm.

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Figure 6: Expression of trpm5 in taste bud cells during zebrafish development. A-C: lateral (A) and ventral (B-C) whole-mount views of larvae 3 dpf; D-F: lateral (D) and ventral (E-F) whole-mount views of larvae staged 5 dpf, with anterior to the left in all pictures. Scale bars of 100 µm are indicated in A for all pictures shown. Abbreviations: PTB = pharynx taste bud; GTB = gill taste bud.

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Figure 7: Expression of trpm7 in whole-mount zebrafish during embryonic and larval stages. A-C: dorsal (A), lateral (B), and ventral (C) views of embryos 24 hpf; D-F: lateral views of embryos staged 48 hpf; G-J: 3 dpf larvae shown from lateral (G) and ventral (H-J) sides, respectively; K-M: lateral (K) and ventral (L-M) views of larvae staged 5 dpf. Zebrafish are orientated with anterior to the left. Arrowheads in E, G, and H mark expression domains in unknown anatomical structures. Scale bars of 100 µm are indicated in the first picture of a series until changed. Abbreviations: L = lens; PST = proximal straight tubule; Ic = ionocyte; PCT = proximal convoluted tubule.