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Citation: Silic, M.R.; Zhang, G.
Bioelectricity in Developmental
Patterning and Size Control:
Evidence and Genetically Encoded
Tools in the Zebrafish Model. Cells
2023,12, 1148. https://doi.org/
10.3390/cells12081148
Academic Editors: Natascia Tiso and
Francesco Argenton
Received: 4 March 2023
Revised: 3 April 2023
Accepted: 10 April 2023
Published: 13 April 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
cells
Review
Bioelectricity in Developmental Patterning and Size Control:
Evidence and Genetically Encoded Tools in the Zebrafish Model
Martin R. Silic 1and GuangJun Zhang 1,2,3,4,*
1Department of Comparative Pathobiology, Purdue University, West Lafayette, IN 47907, USA
2Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA
3Purdue Institute for Inflammation, Immunology and Infectious Diseases (PI4D), Purdue University,
West Lafayette, IN 47907, USA
4Purdue Institute for Integrative Neuroscience, Purdue University, 625 Harrison Street,
West Lafayette, IN 47907, USA
*Correspondence: gjzhang@purdue.edu; Tel.: +1-765-496-1523; Fax: +1-765-494-9830
Abstract:
Developmental patterning is essential for regulating cellular events such as axial patterning,
segmentation, tissue formation, and organ size determination during embryogenesis. Understanding
the patterning mechanisms remains a central challenge and fundamental interest in developmental
biology. Ion-channel-regulated bioelectric signals have emerged as a player of the patterning mecha-
nism, which may interact with morphogens. Evidence from multiple model organisms reveals the
roles of bioelectricity in embryonic development, regeneration, and cancers. The Zebrafish model
is the second most used vertebrate model, next to the mouse model. The zebrafish model has great
potential for elucidating the functions of bioelectricity due to many advantages such as external
development, transparent early embryogenesis, and tractable genetics. Here, we review genetic
evidence from zebrafish mutants with fin-size and pigment changes related to ion channels and
bioelectricity. In addition, we review the cell membrane voltage reporting and chemogenetic tools
that have already been used or have great potential to be implemented in zebrafish models. Finally,
new perspectives and opportunities for bioelectricity research with zebrafish are discussed.
Keywords:
zebrafish; patterning; embryonic development; long fin; short fin; pigment; ion channels;
bioelectricity; GEVI; optogenetics; chemogenetics
1. Introduction
Embryonic development is a self-autonomous and robust process in which a new
body develops from a fertilized egg. This developmental process requires coordinated and
complex cellular events such as proliferation, differentiation, and movement. The related
patterning mechanisms are essential and instructive elements that eventually guide the
body shape and organ sizes [
1
–
3
]. The morphogen gradient and transcription network
are the mainstay theories and have been verified in many organ systems of various organ-
isms [
4
–
6
]. Recent and past evidence revealed that ion-channel-related bioelectricity is a
new component of the regulating mechanism for developmental patterning, regeneration,
and cancers [7,8].
Bioelectricity is defined as endogenous electrical signaling across cell membranes
and is mediated by the dynamic distribution of charged molecules [
7
–
13
]. This is repre-
sented by a difference in the net charge of cations and anions inside versus outside a cell.
Many components are involved in electrical potential formation [
12
,
14
]. In essence, the
semipermeable lipid-based plasma membrane acts as an electrical insulator, but also as a
capacitor that can accumulate charge, while specialized passages (ion channels, pumps,
connexins/gap junctions, and solute carriers) regulate ion flow from one side to the other,
altering the voltage of the cell (Figure 1A). All cell types form ionic gradients across their
cell membranes because channels exist throughout all living organisms in all domains of
Cells 2023,12, 1148. https://doi.org/10.3390/cells12081148 https://www.mdpi.com/journal/cells
Cells 2023,12, 1148 2 of 31
life, including plants, fungi, and bacteria [
14
–
24
]. Thus, ion regulation and the resulting
bioelectricity are considered essential properties of living cells across evolution, and their
innate properties can be used for cellular communication [
25
,
26
]. Therefore, understand-
ing additional aspects of bioelectricity in cells and organisms is fundamental for modern
physiology and ontology.
Cells 2023, 12, x FOR PEER REVIEW 3 of 33
Figure 1. Cell membrane potential formation and comparison of neuromuscular excitable cells and
non-excitable somatic cells. (A). Illustration of resting membrane potential, ion regulators, and ionic
concentrations when the cell is in a non-excitable state. Dierent shapes represent various ion reg-
ulators on a cell membrane (blue region). The arrows indicate the movement of ions when the reg-
ulators are open. (B). Comparison of neuromuscular excitable and non-excitable somatic cells. Ex-
citable cells usually exhibit action potentials, while the non-excitable somatic cells have membrane
potential uctuations, which vary in their amplitudes and frequencies.
2. Cellular Contributors to Membrane Potential and Bioelectricity
2.1. Cell Membrane Potential and Concentration Gradients
Bioelectricity can be exhibited in several dierent forms in multicellular organisms:
on cellular, tissue, and organ levels. For example, cell membrane potential or membrane
voltage (Vm) is one of the integral cellular bioelectric properties (Figure 1A). Many essen-
tial cellular physiological processes rely on Vm. These include cross-membrane transport
(e.g., nutrients, salts, water), cell volume control, secretion, the cell cycle, and migration
[13,25]. Additionally, Vm allows for cognitive and motor function through neuronal sig-
naling, resulting in organismal, tissue, or cellular sensory detection, and locomotive
movement [25].
In typical neuronal signaling, the steady-state baseline voltage is called resting Vm,
whereas the excited “signaling” state is called an action potential (AP). The resting cell
membrane potential is the overall combination of ions for a cell, but the equilibrium po-
tential for each ion is dierent in dierent cell types [25], resulting in a range of resting
membrane potentials in each cell type (Table 1). Although this generally results in a range
between −30 and −80 mV, Vm can even exceed a range of −5 mV to −150 mV, depending
on cell type [9]. These resting Vm values can uctuate in a small or large deviation. Large
and rapid depolarization changes from negative to more positive membrane potential are
Figure 1.
Cell membrane potential formation and comparison of neuromuscular excitable cells and
non-excitable somatic cells. (
A
). Illustration of resting membrane potential, ion regulators, and
ionic concentrations when the cell is in a non-excitable state. Different shapes represent various ion
regulators on a cell membrane (blue region). The arrows indicate the movement of ions when the
regulators are open. (
B
). Comparison of neuromuscular excitable and non-excitable somatic cells.
Excitable cells usually exhibit action potentials, while the non-excitable somatic cells have membrane
potential fluctuations, which vary in their amplitudes and frequencies.
Neuronal and muscular systems have been well investigated for their bioelectric
activities. The field of neuromuscular bioelectricity has a relatively long and diverse
history [
27
]. Luigi Galvani first demonstrated the relationship between electricity and
animals in 1780 by electrically stimulating frog limbs to cause movement. However, it
was almost another hundred years before the first measurements of action potentials, in
1865 by Julius Bernstein, using a differential rheotome [
28
]. The first intracellular electrical
measurements of the resting membrane in the protozoon Paramecium were performed in
1934 [
29
]. Afterward, ion discoveries on neuronal bioelectricity were made by Hodgkin
and Katz, using the giant squid axon as an experimental model [
30
]. Their intracellular
Cells 2023,12, 1148 3 of 31
recording studies paved the way for neurology and the fundamental understanding of
action potentials [
31
]. One example is the combinational uses of neuronal axons’ action
potential, voltage-gated Ca
2+
ion channels, and synaptic neurotransmitters for neural
signals [
32
]. However, the function of bioelectricity remains largely unknown outside
of a neuromuscular context. Expanding on these concepts of neuronal bioelectricity and
neurotransmitters, it is not inconceivable that other electrical signals could travel across the
membranes of non-nerve cells and trigger various responses: to cause other ions to enter
the cell (or be released from internal stores); to change transcriptional regulation of the
machinery; to cause protein modifications, such as conformation or phosphorylation, to
affect function; as well as to modify plasma membrane molecules such as receptors, kinases,
and lipids [
33
–
36
]. Indeed, recent advances in bioelectricity research regarding embryonic
development, regeneration, cancers, and potential mechanisms have been systematically
reviewed [
8
,
37
,
38
]. In this review, we will focus on zebrafish mutants with patterning
defects and genetic tools that are related to bioelectricity.
2. Cellular Contributors to Membrane Potential and Bioelectricity
2.1. Cell Membrane Potential and Concentration Gradients
Bioelectricity can be exhibited in several different forms in multicellular organisms:
on cellular, tissue, and organ levels. For example, cell membrane potential or membrane
voltage (Vm) is one of the integral cellular bioelectric properties (Figure 1A). Many essential
cellular physiological processes rely on Vm. These include cross-membrane transport (e.g.,
nutrients, salts, water), cell volume control, secretion, the cell cycle, and migration [13,25].
Additionally, Vm allows for cognitive and motor function through neuronal signaling,
resulting in organismal, tissue, or cellular sensory detection, and locomotive movement [
25
].
In typical neuronal signaling, the steady-state baseline voltage is called resting Vm,
whereas the excited “signaling” state is called an action potential (AP). The resting cell
membrane potential is the overall combination of ions for a cell, but the equilibrium
potential for each ion is different in different cell types [
25
], resulting in a range of resting
membrane potentials in each cell type (Table 1). Although this generally results in a range
between
−
30 and
−
80 mV, Vm can even exceed a range of
−
5 mV to
−
150 mV, depending
on cell type [
9
]. These resting Vm values can fluctuate in a small or large deviation. Large
and rapid depolarization changes from negative to more positive membrane potential are
referred to as APs, which are barely reported outside of neuronal and muscular tissues.
These APs are triggered by ion channels that respond to changes in voltage that reach
a certain threshold. More specifically, depolarizations may merge along a neuron axon
or dendrite, eventually pass the Vm threshold for voltage-gated ion channels, and form
an AP [
39
]. These AP waves can propagate from multiple locations, and if two meet
from opposing directions, they will annihilate each other [
40
]. This quick (millisecond)
and extreme (
≥
100 mV difference) swing in voltage, caused by altering intracellular ion
concentrations, is unique to excitatory cells. However, increasing evidence shows smaller
and longer-duration types of electrical signaling events in other, non-excitable cell types,
such as melanocytes, can have significant effects [
41
]. Changes in Vm of non-excitable
somatic cells could come from a variety of factors and would not be classified as traditional
AP signals (Figure 1B). Smaller and less extreme increases or decreases in Vm can occur
within embryonic neural and non-neural tissues over various periods, such as milliseconds,
seconds, minutes, hours, or even days. Such subtle bioelectric signals may be essential in
cell differentiation and embryonic patterning during development [7–9,37].
Cells 2023,12, 1148 4 of 31
Table 1. Reported cellular membrane potential of common vertebrate cell types.
Somatic Cells Embryonic Origin Millivolts (mV) References
Skeletal myocyte Paraxial mesoderm From −91 to −65 [42]
Heart myocyte Lateral plate mesoderm From −95 to −40 [43]
Gut smooth muscle myocyte Lateral plate mesoderm From −70 to −35 [44]
Gliocyte Neuroectoderm or neural crest About −80 [45]
Neuron Neuroectoderm From −85 to −65 [46,47]
Adrenal cortex Intermediate mesoderm From −71 to −66 [48]
Adrenal medulla Neural crest From −32 to −20 [48]
Lymphocyte Mesoderm From −70 to −50 [49]
Thyroid follicular cell Foregut endoderm From −70 to −60 [50]
Chondrocyte Mesoderm and neural crest From −64 to −48 [51]
Fibroblast All three embryological germ layers From −25 to −16 [52,53]
Liver hepatocyte Ventral foregut endoderm From −50 to −20 [54]
Pancreas β-cell Foregut endoderm From −80 to −60 [35]
Epithelial cell All three embryological germ layers From −70 to −20 [55]
Melanocyte Neural crest From −50 to −40 [56–58]
White adipocyte Mesoderm and neuroectoderm From −69 to −17 [59]
Osteocytes Mesoderm and neural crest About −60 [60]
Cancer and tumor cells All three embryological germ layers From −50 to −5 [10,56,61,62]
The electromagnetic force of the differential distribution of ions across the cell mem-
brane generates the electric potential. Thus, the concentration gradient of each ion molecule
jointly contributes to Vm value [
63
]. For example, there is a high level of potassium (K
+
)
and low levels of sodium (Na
+
) within cells at resting Vm. High levels of intracellular K
+
and extracellular Na
+
ions are mainly established by the sodium/potassium ATPase pump
(Figure 1A). One ATPase pump binds three intracellular Na
+
ions, utilizes ATP to change
conformation via phosphorylation, and releases the three Na
+
ions into the extracellular
space. Next, two extracellular K
+
ions will bind to this outward-facing conformation,
causing dephosphorylation and reversal of conformation that allows potassium ions into
the cell against its concentration gradient [
7
,
64
]. This form of active transportation and the
resulting electrochemical gradient is responsible for high intracellular potassium.
The electrical potential difference that counteracts or balances the concentration gradi-
ent for a given ion is called equilibrium potential. If only one permeant ion species exists
in a cell, its resting membrane potential will equal the equilibrium potential for that ion.
Potassium and sodium ions are the two main contributors to membrane potential, but
Cl
−
and Ca
2+
ions can also affect Vm, in addition to other charged molecules, such as
protons (H
+
) and organic anions, depending on cell types. Generally, potassium equilib-
rium potential is close to resting cell membrane potential in many cell types, including glia
and neurons. Thus, maintenance of high intracellular potassium is critical for establishing
resting Vm [
32
,
63
]. This difference in concentration is hard to maintain, and potassium
ions can exit the cell through various leak channels, such as K2P potassium channels on the
plasma membrane [
65
]. Removing positively charged K
+
ions from the cell will result in a
more negative electrical charge, forcing more positive ions to be pulled back into the cell
against the chemical gradient. This constant cycling of potassium being pumped into cells
and leaking out helps to establish the electric potential of resting Vm. Eventually, these
electric and gradient forces will reach equilibrium. This balance can be mathematically
described in the Nernst equation [32,63].
Cells 2023,12, 1148 5 of 31
2.2. Membrane Potential Contributors: Ion Channels, Gap Junctions, and Solute Carriers
Ion channels are a group of transmembrane proteins that significantly contribute to
overall cellular bioelectricity. Channels are essentially small pores in the cell membrane
that alter permeability for specific ions based on selectivity (molecular charge and size) and
gating (what is required to open the channel) [
66
]. Channel conductivity is aligned with
the ion concentration gradient, so energy is not required for a high rate of ion-selective
transport. However, the channels will only allow ions to flow down their concentration
gradient (moving from high to low concentration areas). The composition of these channels
on the cell membrane has been compared to an electronic component called a field-effect
transistor [
67
]. In the human genome, more than 400 family members of ion channels are
currently characterized, accounting for around 1.5% of the genome [
68
]. A comprehen-
sive list of human ion channel details can be found on the HUGO Gene Nomenclature
Committee website and the IUPHAR/BPS Guide to Pharmacology [66,69].
Based on ion selectivity, ion channels can be classified as sodium (Na
+
), calcium (Ca
2+
),
potassium (K
+
), chloride (Cl
−
), or proton (H
+
) channels, as well as non-charged molecules
such as aquaporins [
66
,
70
]. The most direct Vm-contributing ion channels are K
+
and
Na+, while the others play a minor role or secondary messenger role, such as that of Ca2+.
Each ion channel type can then be further categorized by gating mechanism. One group,
voltage-gated channels, will open or close when their voltage-sensitive domains detect a
specific change in membrane potential, usually a significant depolarization from action
potentials in neurons. Another type, ligand-gated ion channels, relies on their receptor
binding a particular ligand to cause or prevent ionic flow. A third category, leak channels,
continually allows a small amount of sodium or potassium to leave the cell, regardless of
Vm state [
65
,
71
,
72
]. This type of channel can profoundly impact Vm because it can heavily
affect the ion gradient at different stages of excitatory conditions. There are additional
mechanisms to regulate or gate leak channels, such as temperature, mechanical force,
and light [
65
,
71
,
72
]. Another interesting group of channels is that of inwardly rectifying
potassium channels (Kir) [
73
]. These channels allow K+ ions to move more easily into,
rather than out of, a cell when the cell membrane is depolarized. This is because the
intracellular concentration of potassium is so high at rest, and this type of ion movement
occurs against the concentration gradient. Even when these are functioning, it is difficult
for K+ ions to enter the cell, and they might leak out. Due to this unique characteristic,
the Kir channels will impact concentration gradients, resting membrane potential, and cell
excitability [
73
]. Furthermore, different channels can show distinct levels of rectification
(e.g., high or low). The lipid species, such as PIP2 (phosphatidylinositol 4,5-bisphosphate),
can further regulate Kir channels, as can Mg
2+
, polyamines, phosphorylation, or protein–
protein interactions [73].
Gap junctions are membrane proteins that physically connect adjacent cells to allow
ions, small molecules, and electrical impulses to pass directly by a regulated gate between
cells. Like ion channels, their conductance is passive and down an electrochemical gradient.
Thus, they do not rely on ATP-like ion pumps. Gap junctions are formed by connecting
proteins called connexins and pannexins in vertebrates and innexins in invertebrates (de-
pending on the number of Cys residues in their extracellular loop and glycosylation) [
74
].
These connexins have unique protein structures, properties for permeability, and gating.
Each gap junction comprises six connexin subunits on one cell that oligomerize with another
six connexins on an adjacent cell. The connection of the same connexin isoform is called
homogenous/homomeric, but these properties can change and become more complex by
forming heterogeneous/heteromeric gap junctions [
75
]. When these connexins are not
coupled to form a gap junction, they are known as hemichannels [
76
]. These hemichannels
may serve as an ionic and molecular interchange routes between the cytoplasm and the
extracellular environment [
77
]. Gap junctions and hemichannels play significant roles in
cell-to-cell communication by exchanging ions, small molecules, subcellular vesicles, elec-
tric impulses, and organelles, due to their relatively larger pores [
78
]. Thus, they are natural
modulators of cellular bioelectricity. Electrical synapses between neurons can be considered
Cells 2023,12, 1148 6 of 31
a specialized gap junction. In addition, gap junctions have also been found to be needed
for direct cell communication in tunneling nanotubules
(TNTs) [79,80].
Gap junctions are
crucial for many physiological processes, including synchronized depolarization of car-
diac muscle and embryonic development [
81
,
82
]. One ubiquitous gap junction, connexin
43 (CX43), has been implicated in multiple organisms and diseases and it contributes to
electrical signaling [
83
]. Connexin mutations and misregulations have been shown to cause
many diseases, such as neurodegenerative diseases and congenital morphological defects
in mice and humans [84,85].
Another group of Vm ion regulators is solute carrier proteins (SLCs). These proteins
utilize secondary active transport, where thermodynamically favorable reactions (i.e., ions
moving down their concentration gradient) are paired with one or more other molecules to
be transported in an unfavorable reaction [
86
]. The free energy provided by the movement
in the favorable direction makes movement in the less favorable direction possible and
allows transport without directly consuming cellular energy. These reactions utilizing
the electrochemical gradient can occur with both substrates moving in the same direction,
known as symporters, or substrates moving in opposite directions, known as antiporters.
Thus far, over 450 transporter proteins are found in the plasma membrane of cells and
subcellular organelles [
86
–
88
]. These SLCs have an extensive range of substrate specificity,
including ions, organic ions, sugars, vitamins, amino acids, nucleotides, oligopeptides,
drugs, and metals. In addition, some SLCs can transport multiple different biomolecules,
others can only transport a single biomolecule, and up to 30% are “orphan” proteins, whose
substrates remain unknown. Thus, these SLCs have been involved in many physiological
regulations, such as selective barriers, neurotransmitters, nutrition, and metabolic regula-
tion [
86
,
88
]. More than 190 diseases have been linked to SLCs, such as thyroid, hearing,
neurological, metabolic, and congenital defects [
86
,
88
]. Due to the nature of their substrates,
the SLCs could be an essential contributor to cellular bioelectricity.
3. Bioelectricity Evidence from Zebrafish Genetics
3.1. Zebrafish as a Superior Model for Bioelectric Research
The zebrafish has become one of the leading model organisms used in research since
its debut in the 1970s, due to its unique advantages [
89
–
91
]. First, zebrafish share ver-
tebrate biology with humans. Zebrafish possess 70% orthologous genes to humans [
92
].
Second, it is a relatively affordable model, compared to murine models. Third, small
body size and external development make zebrafish embryos an ideal
in vivo
system.
Fourth, tractable genetics has been developed in zebrafish, including large-scale forward
genetic mutagenesis, CRISPR-based reverse genetics, and Tol2 transposon-based trans-
genesis [
93
–
95
]. Furthermore, a significant source of mutation lines is available through
the repository ZFIN, and the greater zebrafish research community is highly collabora-
tive [
96
,
97
]. All these advantages make zebrafish popular for studying developmental
biology, neuroscience, physiology, toxicology, drug screens, and many human diseases
such as cancers [
89
–
91
,
98
,
99
]. Zebrafish are also particularly suited to bioelectric research.
The combination of excellent and well-established genetic tools with transparent external
embryonic development can allow for manageable mutant generation and cutting-edge
microscopy to explore previously unattainable information. These attributes can also be
useful in bioelectric research. Below, we highlight bioelectric-related zebrafish studies that
demonstrate the importance of this model as an optimal way to characterize and uncover
the as-yet-undetermined bioelectric characteristics and mechanistic properties.
3.2. Zebrafish Mutants with Adult Fin-Size Change
Zebrafish adults have two sets of fins: paired fins (pectoral and pelvic) and unpaired
median fins (dorsal, anal, and caudal), aligning their anterior to posterior body mar-
gins [
100
–
102
]. Each fin comprises endoskeletons and external dermal bones: the fin rays,
or lepidotrichs. The adult fin size and its proportion to the body are generally unvarying.
Zebrafish paired fin development was reported to share similar mechanisms with tetrapod
Cells 2023,12, 1148 7 of 31
limbs, as corresponding signaling centers such as ZPA (zone of polarization) and AER
(apical ectodermal ridge) were characterized in zebrafish [
100
,
103
,
104
]. Although direct
evidence of bioelectricity in zebrafish fin development is still lacking, indirect evidence
came from several zebrafish fin mutants from large-scale forward genetic screenings. These
mutants display either elongated or short fins, and underlying mutated genes are involved
in normal ion regulation via channel, solute carrier, or connexin (Table 2), indicating
that bioelectricity, not a specific ion regulator, is the key to zebrafish fin patterning and
size regulation.
The first reported zebrafish mutant with elongated fin size is longfin(lof
t2
), which is a
dominant mutant that occurred in nature and is present in the widely used Tüpfel fish line.
The causal mutant gene of the lof has remained unknown for decades until recently. Two in-
dependent reports pinpointed Kcnh2a, a voltage-gated potassium channel [
105
,
106
]. There
is a 0.9 Mb chromosomal reversion upstream of the kcnh2a gene on
chromosome 2 [106].
This inversion disrupts gene regulation and causes a change of the cis-ectopic expression
of kcnh2a in zebrafish fins. Similar to the lof
t2
, another longfin (alf
dty86d
), an ENU-induced
mutant, possesses elongated fins in adults in a dominant way [
107
]. This alf
dty86d
mu-
tant was reported to be caused by gain-of-function mutations in kcnk5b, a potassium leak
channel gene [
107
]. The authors also reported larva fish overgrowth and cellular volt-
age change, indicating the Kcnk5b-mediated bioelectricity of fin anlagen could be the
underlying mechanism through local overgrowth [107].
The schleier is another zebrafish mutant with elongated fins. This mutation is caused by
the inactivation of a potassium–chloride cotransporter, slc12a7a/kcc4a [
108
]. This mutant is
also genetically dominant, and homozygous adults exhibit broken stripes and pigmentation
alternations. A CRISPR mutation experiment revealed that the function levels of Kcc4a
correspond to the fin and barbel lengths. In addition, kcnk5b knockout in the schleier
fish embryos can reduce the adult fin lengths, suggesting that Slc12a7a might function
together with Kcnk5, and both might be required for bioelectric regulation in wildtype fish.
Interestingly, the same research group also identified slc43a2/lat4a, an L-leucine amino acid
transporter that can modify the kcnh2a mutation effect in lof
t2
mutant fish, resulting in a
flying-fish-like phenotype [
106
]. This lat4a mutant, lat4a
nr21
, is also dominant and exhibits
a short-finned phenotype in heterozygotes. The interactions between Lat4a and Kcnh2a
in the flying-fish-like zebrafish suggest they are also involved in bioelectric regulation.
Along with this short-finned phenotype, two additional mutants were reported. They
are the shortfin (sof ) mutants (4 alleles: sof
b123
(spontaneous), sof
j7e1
, sof
j7e2
, sof
j7e3
(ENU-
induced)) caused by a hypomorphic mutation in the gap junction, Cx43 [109], as well as a
fish mutant, mau, caused by a dominant missense mutation in aqp3a (aquaporin 3a) [
110
].
Like the lat4a
nr21
mutation, the cx43 mutation in sof also reverted the lof
t2
long-finned
phenotype [
111
], suggesting that Cx43 is another bioelectric regulator for zebrafish fin size.
Our laboratory recently characterized a dominant long-finned mutant, Dhi2059, which
was generated via a large-scale insertional mutagenesis [
112
]. The kcnj13 gene’s exon 5
was disrupted by a retroviral insertion. Although this exon encodes 5
0
UTR (untranslated
region), not protein, viral DNA insertion leads to a transient and ectopic expression of
kcnj13 in the somites between 15S (15-somite stage) and 48 dpf (days post fertilization) in
Dhi2059 fish embryos. Transgenic fish Tg (
−
5.4k pax3a:kcnj13-IRES-EGFP), in which the
kcnj13 gene is under the control of the
−
5.4k pax3a promoter, can phenocopy the long-fin
phenotype. Thus, kcnj13 misregulation resulted in elongated fins in the adult zebrafish,
mainly by increasing the length of fin rays [
112
]. Different from the previously mentioned
long-finned mutants (lof, alf, schleier), our results suggest that the adult fin size can be
determined at the somite stage in early fish embryos. This indicates that bioelectricity
is set up early and could serve as a memory for patterning and size regulation in later
ontology (see detailed discussion in the prospective section). In addition, we showed
that transient expression of multiple potassium channels (kcnj1b, kcnj10a, kcnk9, human
KCNJ13) in zebrafish early embryos (by microinjection) could also cause chimeric long fins
Cells 2023,12, 1148 8 of 31
in injected adult fish. This result suggests it is not a specific potassium channel, but that
bioelectricity is the key to the elongated fin phenotype.
Multiple key points can be obtained by comparing these zebrafish mutants. First, all
the mutant genes are involved in ion regulation, which is intrinsically linked to bioelectricity.
These ion regulators have their own ion type selecting properties and conductance. It
becomes challenging to explain the fin phenotype with a specific channel or ion. Instead,
it is more reasonable that electric signaling is the underlying mechanism. Different ion
regulators with other properties can be used to construct and modify the bioelectric state
of cell groups and tissues. Second, all of these mutations are genetically dominant; most
are gain-of-function, ectopically expressed, or neomorphic. Lastly, the specificity of the
zebrafish fin-size phenotype may be caused by the spatiotemporal distribution of these ion
regulators during embryonic development, as exampled by the Dhi2059 mutant. Taken
together, the zebrafish’s adult fin size could be regulated at multiple stages. Although most
studies reported altered gene expression in fin anlagen or local fins, our experimental data
suggested that somites, the embryonic origin of fin ray progenitor cells, can play a critical
patterning role.
Consistent with zebrafish mutants, it is also worth noting that different potassium
channels were recently identified in other teleosts through genome association studies.
The inwardly rectifying channel gene kcnj15 was mapped to long-finned betta fish [
113
].
Additionally, the ether-à-go-go (EAG) potassium channel gene, kcnh8, was found to be
highly expressed in the male caudal fins in Xiphophorus [
114
]. Like zebrafish, kcnk5bS
was identified as a candidate for long-tailed goldfish [
115
]. Together with zebrafish mu-
tants, these data suggest that ion-channel-mediated bioelectricity plays an essential role in
fin patterning.
Table 2. Zebrafish, published fin-size mutants.
Mutant Fin Gene Mutation
Nature
Fin Ray
Segment
Length
Fin Ray
Numbers
Dominant or
Recessive
Somite or
Local Fin References
lof Long kcnh2a
Ectopic by
cis-regulatory
change
Normal Increased Dominant Local [105,106]
alf Long kcnk5b GOF Increased Decreased Dominant Local [107]
schleier Long
slc12a7a/kcc4a
LOF or dominant
negative?
Dose dependent
Normal Increased Dominant Local [108]
Dhi2059
Long kcnj13
Ectopic by
cis-regulatory
change, Dose
dependent
Increased Decreased Dominant Somite [112]
sof Short cx43 Hypomorphic Decreased Decreased Dominant Local [109]
mau Short aqp3a Neomorphic,
Dose dependent Normal Decreased Dominant Local [110]
nr21 Short slc43a2/lat4a GOF Decreased Not
reported Dominant Local [111]
Note: GOF: gain-of-function; LOF: loss-of-function.
3.3. Zebrafish Mutants with Adult Pigmentation Pattern Alterations
Zebrafish adults exhibit distinct stereotypical stripe patterns along their bodies, with
alternating rows of melanophores (dark pigments) and xanthophores (red-orange pigments)
mixed with iridophores (iridescent pigments) [
116
–
118
]. Local and long-range interactions
and communication among these different pigment cells during embryonic and larval
stages are essential to forming the stripe patterns [
79
,
115
,
116
]. Among many mutant ze-
Cells 2023,12, 1148 9 of 31
brafish lines with altered pigmentation patterns, several are mutations of ion regulators,
suggesting that ion-channel-mediated bioelectric signals play important roles in pigmenta-
tion patterning. Two of the fish mutants, albino and golden, resulted from the loss of function
of solute carrier genes, slc45a2 and slc24a5, respectively [
119
–
121
]. The phenotypic results
of the two mutants are a complete loss of melanophores and light stripes (melanophores
with small and fewer melanin granules), respectively. The two genes are expressed in ze-
brafish melanophores, and light pigmentation was thought to be mainly caused by reduced
melanogenesis due to ion and proton alteration in the melanophores [
120
,
121
]. The trans-
parent (tra) fish possess fewer iridophores, melanophores, and dark spots, instead of stripes,
in adults. This tra is a loss-of-function mutation of the mpv17 gene, which encodes a non-
selective channel that modulates mitochondria membrane potential [
122
,
123
]. Although
the loss of Mpv17 was found to cause a reduction in the number of mitochondria and
reduced pyrimidine synthesis [
123
], the bioelectricity of iridophores might also contribute
to patterning defects.
In addition to chromophore defects, zebrafish stripe patterns were found to be altered
in additional mutants. The leopard (leo
t1
) mutation, also known as tup, is a spontaneous
recessive mutation causing spots in the adult Tüpfel fish line. This mutation is caused by the
cx41.8 (connexin 41.8) gene, which encodes Gja5b in zebrafish [
124
]. Similarly, luchs (luc
tXA9
)
is a mutation of the cx39.4 (connexin 39.4) gene, which encodes Gja4 [
124
]. Both cx41.8 and
cx39.4 are required for melanophore and xanthophore development. Both mutants show
aggregated dark spot patterns instead of stripes. Interestingly, it was shown that these two
connexins could form heteromeric, in addition to homomeric, gap junctions, which are
essential for melanophore and xanthophore cellular communication [
124
,
125
]. Recently,
another mutant zebrafish, schleier, was reported to be caused by hypomorphic function
of another solute carrier, slc12a7a/kcc4a [
108
]. The homozygous mutant fish show broken
stripes in the ventral body flank and anal and caudal fins. Gap junctions usually conduct
small molecules and ions between neighboring cells. Thus, they can modulate molecular
and electrical coupling among the adjacent cells [
81
,
82
], and over longer distances [
79
,
80
].
Additionally, the obelix(obe)/jaguar(jag) mutants, which are caused by a kcnj13 loss of func-
tion, have fewer stripes compared to wildtype fish [
126
]. Kcnj13 is an inwardly rectifying
potassium channel that regulates cell excitability and membrane potential. Based on the
less severe pigmentation phenotype of the kcnj13 null mutants (kcnj13
pu107
, kcnj13
pu109
) our
lab generated, the original alleles (jag
b230
, obetc
271d
, and obe
td15
) are most likely dominant
negative [
112
,
126
]. More recently, kcnj13 expression was found to underlie the pattern
diversification among Danio species via the kcnj13 regulatory changes [
127
]. This potassium
channel gene is expressed in melanophores during development, suggesting that it may
regulate melanophore bioelectric properties. Indeed, cellular electrical communication was
partially disrupted in this mutant. The dissociated melanophores of jag are more depo-
larized when measured with a voltage-sensitive dye, DiBAC4(3), than the melanophores
from wildtype fish. Wildtype melanophores are transiently depolarized when contacted
by the dendrites of a xanthophore, and then moved away from the xanthophore. In con-
trast, jag
b230
melanophores lost contact-dependent depolarizations and repulsive migration
behavior [128].
Three additional zebrafish mutants could also be related to bioelectric regulation,
though the related genes are not direct ion regulators. Spermidine is an endogenous
polyamine that can regulate ion channels and connexins [
129
,
130
]. The idefix (ide
t26743
)
fish is a loss-of-function mutant of the srm (spermidine synthase) gene [
131
]. Homozygous
ide
t26743
mutants have fewer narrowed and often interrupted dark stripes in the trunk and
fewer strips in the fins. This ide mutation can further reduce melanophores when crossed
with leo
t1
,luc
tXA9
, and obe
271d
mutants, suggesting that spermidine may modulate connexin
and potassium channel functions. Moreover, ectopic expression of spermidine/spermine
N1-acetyltransferase (Ssat), a polyamine metabolic enzyme in melanophore, caused broken
stripes and a loss of melanophores in the leo
t1/t1
background, also supporting this idea [
132
].
Another zebrafish mutant, schachbrett (sbr
tnh009b
), is caused by a loss of function mutation
Cells 2023,12, 1148 10 of 31
of tight junction protein 1a (Tjp1a), which is expressed in iridophore [
133
]. Like ide
t1
, the
sbr
tnh009b
mutant exhibits more substantial pigment patterning defects in luc
t32241
and leo
t1
background, indicating Tjp1a may interact with connexins. Thus, Tjp1a may indirectly
affect the bioelectricity of chromatophores. The third zebrafish mutant, mau, also possesses
spotted pigments. The underlying gene of the mau mutation is aqp3a, which is mainly ex-
pressed in skin and muscle, but not in chromatophores [
110
]. Transplantation of aqp3a
tVE1/+
blastomere cells into wildtype and Aqp3a
R220Q
in a transgenic experiment revealed that
Aqp3a might indirectly influence chromatophores for pigment patterning. Aqp3a is a
transporter of non-polar solutes such as glycerol, peroxide, and urea, excluding ions [
134
].
Thus, Aqp3a can modulate the ion concentrations related to cellular bioelectricity.
4. Genetically Encoded Tools That Can Be Used for Studying
Developmental Bioelectricity
Functional studies of the bioelectric mechanisms of embryonic developmental pro-
cesses require suitable tools with which to measure endogenous bioelectricity in a real-time
and non-invasive manner and manipulate cell and tissue bioelectricity without compro-
mising whole embryo tissue integrity. With recent significant advances in neuroscience,
various genetically encoded tools were developed to meet these purposes. Genetically
encoded indicators, or biosensors, can allow us to measure cellular membrane potential, ion
concentration, and even metabolites via fluorescence [
135
,
136
]. Additionally, chemogenetic
and optogenetic tools allow us to manipulate cellular bioelectricity, such as membrane
potential, in a precise manner [
137
–
143
]. Although initially developed for studying neurons,
these tools can also be utilized in other research contexts, such as for studying bioelectricity
in embryonic development.
4.1. Measuring Cellular Bioelectricity: Genetically Encoded Voltage Indicators
Cellular electric neural activity can be accurately measured using traditional electrode
measurements, such as the patch clamp method. This method is highly accurate, but is
generally limited to single-cell recordings and invasive to cells [
144
–
146
]. Calcium has
also been widely used to reflect neuronal electric activities. Genetically encoded calcium
indicators, GECIs, have been widely implemented into the zebrafish model for neural
studies [
147
–
149
], cell migration [
150
,
151
], embryogenesis [
152
,
153
], insulin secretion from
pancreatic beta cells [
154
], and many other biological processes [
155
–
158
]. Although
calcium signals mimic electric signals, they are still different due to their additional function
as a secondary messenger in various cell types. Indeed, a difference was reported in
calcium and voltage signals [
159
,
160
]. Genetically encoded voltage indicators (GEVIs) were
invented to directly measure neurons’ electrical activity, complementary to patch clamp
electrophysiology. These non-invasive, endogenous fluorescent biosensors can function
over multiple cells and tissues to provide a collective understanding of real-time bioelectric
activities versus single cells. These are also more advantageous over previously developed
electrochemical dyes due to their increased speed, genetic specificity, higher sensitivity,
and lack of toxic effects [
161
]. The fastest GEVIs have reported rates up to 1 ms [
135
].
Another advantage of GEVIs is the ability to provide results over extended periods. While
these sensors offer several advantages, they do have some drawbacks, such as genetic
modification and the high-end fluorescent microscopes required, variable dynamic ranges,
and signal-to-noise ratios [
135
]. However, with the advance of technical developments,
these shortcomings are being overcome, and GEVI applications are expanding beyond
neuroscience into many fields, such as developmental biology.
Thus far, numerous advancements and variations of GEVIs have been developed (Table 3).
These GEVIs usually fall into one of three categories (Figure 2A) [
135
,
162
,
163
]: (1). GEVIs
based on a voltage-sensitive domain (VSD) within the cell membrane, usually from the tunicate
(Ciona intestinalis) voltage-sensitive phosphatase, PTPE [
164
,
165
]. The VSD can be linked with
either a single fluorescent protein (FP), dual FPs for FRET (Forester resonance energy transfer)
signaling, or even bioluminescence. (2). Opsin-based GEVIs, with and without additionally
Cells 2023,12, 1148 11 of 31
combined FPs to improve brightness. (3). There is a group of hybrid GEVIs that combine
these different components with the addition of brighter and more photostable synthetic
dyes [
135
,
162
,
163
]. Each GEVI has its unique properties and application niche. Many of these
GEVIs have also been examined and utilized in zebrafish research (Table 3).
One commonly used GEVI is ASAP (accelerated sensor of action potentials) based
on VSD design. A circular permutated GFP is inserted int the middle of S3–S4 loop of
the VSD. Thus, when the VSD protein confirmation is altered by Vm, the intensity of GPF
fluorescence will change correspondingly (Figure 2A). When the cell membrane is hyperpo-
larized, the ASAP1 fluorescence signal is brighter. This ASAP1 was successful, and neuron
bioelectricity has been well-documented in many model organisms, including zebrafish. A
few updated versions have also been developed to improve its speed, signal-to-noise ratio,
and sensitivity [
166
–
169
]. Our lab has generated a ubiquitous transgenic reporter zebrafish
line Tg(ubi:ASAP1) [
170
,
171
]. With this ASAP1 transgenic fish line, real-time endogenous
cellular bioelectric activities can be visualized in fish embryos, larvae, adults, and even
tumor tissues [
170
]. Our results are consistent with an independent report on Tg(UAS:
ASAP1), a binary transgenic fish line for tracking larval fish neuronal circuitry within
cerebellum, optic tectum and spinal cord [
172
,
173
]. In addition, we made new observations
in early fish embryos. We found that a transient local membrane depolarization occurs
before cleavage furrow formation during zebrafish embryo cleavage stages (1–64 cells).
This phenomenon is consistent with calcium signaling measured by GCaMP6s [
153
]. These
Vm changes are not static, but dynamic during the cell division period. Moreover, these Vm
dynamic changes are not perfectly synchronized among early cells. These results suggest a
biological function of Vm in cell division. Membrane potential changes have been shown to
influence the organization of phospholipids. These are known as critical components of the
cleavage furrow and cytokinesis [
36
,
174
,
175
]. Once zebrafish embryos enter the blastula
stage, the bioelectric signals become whole-cell transient hyperpolarizations, mainly found
in the rapidly dividing superficial layers of the blastula (EVL) and yolk syncytial layer
(YSL). During gastrulation, Vm transients continued in the EVL and YSL, and started to
occur in the deeper cells. Moreover, we noticed differential Vm among different embry-
onic tissues and somite-specific hyperpolarization events during the zebrafish embryonic
segmentation period. These results demonstrated that the ASAP family and potentially
other GEVIs could be readily used for measuring embryonic bioelectricity. We expect more
bioelectric biology to be revealed by organ- and/or tissue-specific zebrafish transgenic fish
lines. For example, different cell types in the zebrafish fins, pigment cells, and the other
cell types in the skin can be characterized for their physiological bioelectric properties (for
details, see the prospective section).
It is worth noting that GEVIs are not limited to zebrafish. They have also been
applied to other organisms. For example, multiple studies have shown the utility of
genetically encoded indicators in fruit flies, but have, thus far, only been focused on
neuronal-related studies [
167
,
169
,
176
–
178
]. In mice, fewer research studies have utilized
GEVIs
in vivo
, and most of these studies focused on neurological research [
178
–
182
]. In
addition, Xenopus oocytes were used to characterize Arclight, but did not address any
developmental biology [
183
]. Nevertheless, their results showed that these sensors could
be employed to characterize bioelectricity in Xenopus if needed.
Cells 2023,12, 1148 12 of 31
Cells 2023, 12, x FOR PEER REVIEW 12 of 33
Figure 2. Illustrations of design principles of common GEVIs, optogenetic and chemogenetic tools.
(A). Schematic structures of VSD (voltage sensitive domain) based and opsin-based GEVIs. The volt-
age-sensitive domain is labeled brown in the cell membrane. A uorescent protein (FP) is inserted
into the S3–S4 loop. When Vm is altered, the uorescence intensity will change accordingly. The
light-sensitive opsin (dark blue) can sense the Vm of the membrane and act as a chromophore. Thus,
it can be used to measure the Vm with or without a connected FP, which can enhance the overall
signal. (B). Principles of Optogenetics and chemogenetics. The optogenetic tools are based on light-
sensitive channel rhodopsins that conduct protons or chloride. The PSAMs are mutated ligand-
gated ion channels for sodium or chloride. They are controlled by articial PSEM (pharmacologi-
cally selective eector molecules) ligands. In contrast, the DREADDs are mutated ligand-gated
GPCRs (G-protein-coupled receptors). Depending on the type of G protein, they can increase or
decrease Vm via GIRK channels, calcium signaling, and cAMPs. The arrows indicate the movement
of ions when the regulators are open.
One commonly used GEVI is ASAP (accelerated sensor of action potentials) based on
VSD design. A circular permutated GFP is inserted int the middle of S3–S4 loop of the
VSD. Thus, when the VSD protein conrmation is altered by Vm, the intensity of GPF
uorescence will change correspondingly (Figure 2A). When the cell membrane is
Figure 2.
Illustrations of design principles of common GEVIs, optogenetic and chemogenetic tools.
(
A
). Schematic structures of VSD (voltage sensitive domain) based and opsin-based GEVIs. The
voltage-sensitive domain is labeled brown in the cell membrane. A fluorescent protein (FP) is inserted
into the S3–S4 loop. When Vm is altered, the fluorescence intensity will change accordingly. The
light-sensitive opsin (dark blue) can sense the Vm of the membrane and act as a chromophore. Thus,
it can be used to measure the Vm with or without a connected FP, which can enhance the overall
signal. (
B
). Principles of Optogenetics and chemogenetics. The optogenetic tools are based on light-
sensitive channel rhodopsins that conduct protons or chloride. The PSAMs are mutated ligand-gated
ion channels for sodium or chloride. They are controlled by artificial PSEM (pharmacologically
selective effector molecules) ligands. In contrast, the DREADDs are mutated ligand-gated GPCRs
(G-protein-coupled receptors). Depending on the type of G protein, they can increase or decrease Vm
via GIRK channels, calcium signaling, and cAMPs. The arrows indicate the movement of ions when
the regulators are open.
Cells 2023,12, 1148 13 of 31
Table 3. List of published genetically encoded voltage reporters.
GEVIs Fluorescence Indication Fluorophore Tested in Zebrafish References
VSD-based
ASAP1–3 Hyperpolarize—brighter GFP
Whole fish embryos and larva.
Adult malignant nerve sheath
tumors, larval fish cerebellum,
spinal cord.
[166–173]
ASAP4 Depolarize—brighter GFP [184,185]
Marina Depolarize—brighter GFP [186]
FlicR1 Depolarize—brighter RFP [187]
Arclight Hyperpolarize—brighter GFP [188,189]
Bongwoori Hyperpolarize—brighter GFP Larval olfactory bulb [190,191]
VSD-based
Aahn Hyperpolarize—brighter
(external) GFP [192]
VSFP x Depolarize—FRET increase Multiple Larval zebrafish heart [193–200]
Mermaid Depolarize—FRET increase Multiple [201]
Nabi Depolarize—FRET increase UGK/mKO [202]
JEDI-2P Hyperpolarize—brighter GFP [178]
Opsin-based
Arch Depolarize—brighter GFP [203]
QuasAr x Hyperpolarize—brighter Multiple Larval zebrafish heart [204–208]
Archon1 Depolarize—brighter GFP/RFP
Brain and spinal V3 interneurons
[209,210]
Ace x Hyperpolarize—brighter Green/RFP [211,212]
Ace-mNeon2 Hyperpolarize—brighter GFP [182]
VARNAM Hyperpolarize—brighter RFP [213]
VARNAM2 Hyperpolarize—brighter RFP [182]
pAce Depolarize—brighter GFP [182]
pAceR Depolarize—brighter RFP [182]
Dye- or bioluminescence-based
Voltron Hyperpolarize—brighter Multiple dyes Larval brain [214]
Voltron2 Hyperpolarize—brighter Multiple dyes
Larval olfactory sensory neurons
[215]
Positron Depolarize—brighter Multiple dyes Larval zebrafish brain [216]
hVOS Depolarize—brighter Green dye [217]
Voltage spy Depolarize—brighter Green dye [218]
LOTUS Depolarize—FRET increase Blue/green
bioluminescence [219]
AMBER Depolarize—voltage-gated
luciferase increase
Blue/green
bioluminescence [220]
4.2. Manipulate Cellular Bioelectricity: Optogenetic and Chemogenetic Tools
Another requirement to elucidate the bioelectric signaling mystery is the direct and
specific perturbation of the normal electrical state of cells and tissues. As this was a major
task for neuroscience, optogenetic and chemogenetic tools were already developed as
experimental approaches (Figure 2B and Table 4). These tools have, thus far, demonstrated
Cells 2023,12, 1148 14 of 31
the capability to alter cell-specific electrical states of neurons to hyperpolarization and
depolarization, allowing a precise level of control in various organisms [
137
–
139
,
221
–
223
].
Optogenetics: Optogenetics modulates bioelectricity through microbial (type I) opsins,
which are light-sensitive ion pumps and ion channels found in prokaryotic and eukaryotic
microbial organisms [
224
]. These type I opsins can conduct cations or anions under the con-
trol of different wavelengths of light [
225
–
227
]. When exposed to specific wavelengths of
light on expressing cells, channels open to allow specific ions, such as H
+
, Na
+
, Ca
2+
, or Cl
−
,
into cells, resulting in increased or decreased Vm. The use of optogenetics in zebrafish has
been primarily targeted in neuroscience studies. These have been used to modify swimming
behavior [
228
] and locomotion behavior [
229
], perturb hair cell sensory receptors [
230
],
axon guidance control [
231
], and alter olfactory responses [
232
]. Optogenetic tools have
also been used for other zebrafish research fields, such as heart physiology [
233
,
234
], and
melanophore patterning [
235
]. In the zebrafish melanocyte study, ChR2 was expressed in
the melanophores of zebrafish that were then placed in tanks exposed to blue light to stim-
ulate depolarization [
235
]. As a result, these transgenic fish began to lose the boundaries of
their standard stripe patterns [
235
]. Interestingly, this was partially reversed after allowing
the depolarized cells to return to their average membrane potential, suggesting that endoge-
nous bioelectric signals are essential for maintaining pigment homeostasis. This study also
provided direct evidence of bioelectric signals in zebrafish pigment patterning. Recently,
a set of optogenetic transgenic zebrafish lines have been created under the control of the
UAS promoter [
141
]. This will accelerate bioelectric research in zebrafish. Moreover, with
the great success of light-sensitive rhodopsin, new optogenetic tools have been invented
with which to control gene expression, protein localization, and activity, using a new set
of light-sensitive proteins such as phytochromes, blue light using flavin (BLUF) domain
photoactive proteins, cryptochromes (e.g., CRY2-CIB1), and light oxygen voltage (LOV)
domain proteins. Their applications to developmental biology and zebrafish have already
been reviewed [
236
,
237
]. These new optogenetic tools are also utilized for investigating
zebrafish bioelectricity.
Chemogenetics: Chemogenetics is a genetic approach to perturb cellular electrical
activity using synthetic molecules through either mutated G-protein-coupled receptors
(GPCRs) or ligand-gated ion channels that no longer function normally, but only in the
presence of inert molecules [222,223].
DREADDs (Designer receptors exclusively activated by designer drugs) are one of
the mainstream chemogenetic tools that are commonly used in neuroscience, including in
the study of behavior, circuits, and diseases [
222
,
238
–
240
]. DREADDs are designed based
on mutated muscarinic and opioid receptors. Four types (hM3DGq, hM4DGi, hM3DGs,
and KORD) alter cellular Vm through downstream signaling changes, such as the arrestin
pathway, intracellular Ca
2+
, and cAMPs that indirectly modulate ion channels. The original
agonist is clozapine-N-oxide (CNO), a derived metabolite of clozapine, which is used as
an antipsychotic. Thereafter, it was found that CNO can be converted into clozapine and
cause psychoactive side effects in murine models [
241
]. Subsequently, more potent and
specific agonists were invented, including JHU37152, JHU37160, and deschloroclozapine
(DCZ) [
242
,
243
]. In addition to the choice of agonist, the diverse signaling pathways down-
stream of receptors make electricity manipulation less straightforward. Furthermore, the
biological reactions could be variable in different cell types. Recent progress on DREADD
structure activation is helpful for us to understand the mechanisms of their activation [
244
].
Still, careful experimental design was proposed to overcome the two potential issues in
animal models [
245
]. Despite these weaknesses, the DREADD tools have found their
way into various animal models, including flies, mice, rats, and primates [
246
]. In ad-
dition, DREADDs have also been extended to other research fields, including diabetes
and endocrinology [
247
,
248
]. However, they have not been implemented in zebrafish
yet. One study tried to utilize DREADD in zebrafish by microinjections, but failed in the
endeavor [
249
]. Instead, they demonstrated that transient receptor potential (TRP) channels
worked in the zebrafish embryos, and successfully manipulated Rohon–Beard and trigemi-
Cells 2023,12, 1148 15 of 31
nal sensory neurons using islet-1 enhancer-driven TRP channels [
249
]. TRPV1 was activated
by capsaicin, TRPM8 was activated by adding menthol, while TRPA1 activity required
temperatures above 28
◦
C. Activation of TRPs induced dose-dependent locomotion and
ablation, and altered wake-sleep behaviors [
249
]. In addition, the TRPV1-based approach
was verified in another zebrafish study for modulating calcium flux in neutrophil [
150
].
Our laboratory has applied one of the DREADD, hM4DGi, to zebrafish embryos and larvae,
and was able to change melanophore pigment cell dispersion [
250
]. Although the voltage
reporter efficiency still needs to be improved, our results suggest that DREADD can be
applied to zebrafish bioelectricity research.
PSAMs (pharmacologically selective actuator modules) are another set of chemoge-
netic tools. PSAMs share a similar key-and-lock concept, utilizing artificial inert molecules
as agonists. For the actuator, instead of using mutated GPCRs as a lock, PSAMs use ligand-
gated ion channels, such as nicotinic receptors (nAChR), serotonin receptor 3 (5HT3), GABA
receptors, and the glycine receptor (GlyR). Each PSAM has a ligand binding domain (LBD)
and a channel. These are mutated ligand-gated ion channels that can only be activated
by PSEMs (pharmacologically selective effector molecules) [
251
,
252
]. Recently, a ligand-
binding domain was genetically engineered, and more potent agonists were identified.
Thus, this ultrapotent system is helpful for research and suitable for therapeutic appli-
cations, as exampled in mice and monkeys [
251
]. The first generation PSAM-GlyR was
expressed in zebrafish horizontal cells (HCs), which connect rod and cone photoreceptors
via synapses [
253
]. Disrupting Vm of HCs resulted in altered light response and lateral
inhibition in retinal ganglion cells. This study illustrated that the ultrapotent PSAM-PSEM
system could be extended for zebrafish bioelectric research.
In addition to the chemogenetic and optogenetic tools mentioned above, direct genetic
modification, i.e., adding or deleting an ion channel regulator, is also achievable with the
established tol2-transposon transgenic system and CRISPR technology [
254
]. This has
already been demonstrated, such as in the transgenic Tg(
−
5.4k-pax3a:kcnj13-IRES-EGFP),
where transient ectopic expression of kcnj13 in zebrafish dermomyotome causes a long-
finned phenotype in adults [
112
]. Nevertheless, this approach depends on already-known
ion channel regulators and an available tissue-specific promoter. In addition, this genetic
modification cannot be turned on and off, as with chemogenetic and optogenetic tools.
Nevertheless, these tractable genetic tools are critical for implanting GEVIs, chemogenetics,
and optogenetics into zebrafish.
Cells 2023,12, 1148 16 of 31
Table 4. Common optogenetic and chemogenetic tools.
Optogenetic Tools Chemogenetic Tools
Name Activation Method Activation
Result
Tested in
Zebrafish References Name Activation
Method Activation Result Tested in
Zebrafish References
ChR2 Blue light (470 nm) Depolarization
Melanophores,
hair-cells,
neurons
[141,230,235,
255]hM4DGi DREADD
agonists
Hyperpolarization
Melanophore [222,250]
eNpHR3.0 Yellow light (590 nm)
Hyperpolarization
Neurons [141,256] hM3DGq DREADD
agonists Depolarization [222]
CoChR Blue light (470 nm) Depolarization Neurons [141,257] hM3DGs DREADD
agonists Depolarization [222]
GtACR1 Green light (515 nm)
Hyperpolarization
Neurons, heart [141,258–260] KORD DREADD
agonists
Hyperpolarization
[222]
GtACR2 Blue light (470 nm)
Hyperpolarization
Neurons, heart [141,259–261]PSAM-5HT3-
HC PSEM ligands Depolarization Horizontal cells [251]
BLINK2 Blue light (455 nm)
Hyperpolarization
Hair cells,
lateral line
neuromasts,
neurons
[262]PSAM-5HT3-
LC PSEM ligands Depolarization Horizontal cells [251]
CheRiff UV light (460 nm) Depolarization Neurons [141,204] PSAM-GlyR PSEM ligands
Hyperpolarization
Horizontal cells [251,253]
Chronos Yellow light (500 nm) Depolarization Neurons [141,263] TRPV1 Capsaicin Depolarization Neurons,
neutrophils [150,249]
eArchT3.0 Yellow (570 nm)
Hyperpolarization
Neurons [141,264] TRPM8 Menthol Depolarization Neurons [249]
ChrimsonR Red light (590 nm) Depolarization Neurons [141,265] TRPA1 >28 ◦C Depolarization Neurons [249]
GluCl v2.0 Ivermectin hyperpolarization [266]
Cells 2023,12, 1148 17 of 31
5. Prospects and Opportunities: Future Directions for Developmental Bioelectricity
The above-mentioned zebrafish fin and pigment genetic mutations are indirect ev-
idence of bioelectricity in developmental patterning. Direct bioelectricity research in
zebrafish is only possible now because of the recent availabilities of new voltage biosensors
and manipulators. Although, here, we use zebrafish fin size and pigment cell patterns as
examples, many other research directions can be pursued in this field. Below, we propose
four major perspectives.
5.1. Systematic Zebrafish Embryo Bioelectricity Characterization
Though electrical signaling in neuronal tissues has been widely accepted and exten-
sively investigated, other embryonic tissues remain completely unexplored. Characterizing
non-neuronal tissues/cellular bioelectric signaling during embryogenesis is essentially
the first step for deciphering the roles of bioelectricity. The
in vivo
real-time systematic
characterization of vertebrate embryos has only just begun. Our Tg(ubi:ASAP1) fish line
provided the first example of endogenous hyperpolarization signals of embryonic tis-
sues [
171
]. More tissue-specific fish lines can be created with newly developed, more
sensitive GEVIs, such as JEDI-2P, voltron2, Ace-mNeon2, and VARNAM2 using Tol2 trans-
genesis and CRISPR knock-in [
178
,
182
,
215
]. For example, in order to investigate somite
cell contribution to the fins in our Dhi2059 mutant, somite compartments (dermatome,
sclerotome, and syndetome) can be labeled with ASAP1 and other GEVIs to investigate
the bioelectricity of these embryonic tissues in the future. On the other hand, faster and
more sensitive imaging technologies, such as light-sheet fluorescence microscopes, have
made this task more achievable [
171
,
267
,
268
]. It is possible that not only cellular Vm, but
also its fluctuation amplitude, frequency, and rhythms, can serve as cell signals. Such
tissue-specific, cell-type-specific, or subcellular bioelectric imaging in zebrafish embryos
will reveal unprecedented insight into the functions of bioelectricity during embryonic
development. Moreover, this information will be helpful in testing the theoretical concept
of “the bioelectric code” [
269
–
271
]. In addition to GEVIs, biosensors for ions, such as the
potassium sensor GINKO2 [
272
] and the chloride sensor ClopHensor [
273
], can also add
another layer of information on bioelectricity.
5.2. Identifying Bioelectricity Contributing Genes and Redundancy of Ion Regulators
The cellular bioelectricity of a given cell type is composed of many ion regulators, as
discussed above. However, major contributors of each cell type at a given embryonic stage
also remain uncharacterized. Technically, the major challenge is ion regulator redundancy.
Forward genetics starts with mutants created by random mutagenesis, and then addresses
which gene is responsible for the mutant phenotype. This approach is powerful for iden-
tifying critical genes given a phenotype, especially embryonic development, in multiple
model organisms, including flies, worms, mice, and zebrafish [
94
,
274
–
276
]. However, this
approach might miss the underlying genes when genetic or biochemical redundancy exists.
It is already known that multiple ion channels, connexins, pumps, and solute carriers can
contribute to the overall cellular bioelectronic properties, though some are more prominent
than others, given a cell type. There are 817 channel/transporter proteins known in the
human genome in the cell membrane [
277
]. Many transporters underline many cellular
functions, but also create redundancy that could make cellular bioelectric homeostasis very
robust (Figure 3) [
278
]. Thus, it is unsurprising that only a limited number of zebrafish
mutants were identified with altered fin and pigment patterns. Thus far, all the known
fin-size mutations are genetically dominant. Most of them are either ectopically expressed
(kcnj13,kcnh2a) or gain-of-function mutations (kcnk5b, aqp3a, and lat4a) (Table 2). This ismost
likely because the loss of one ion regulator is generally insufficient to cause overall cellular
bioelectric change. However, overexpression or gained function of ion regulators could
drive the cell’s bioelectric property out of its physiological range (Figure 3). Thus, this type
of mutant displays a developmental patterning phenotype. Dominant negative mutations
of ion regulators may also function by interrupting structurally similar proteins in the same
Cells 2023,12, 1148 18 of 31
family. For example, the kcc4a in the schleier mutation could be interpreted as a dominant
negative form, since the null mutant has no fin phenotype [
108
]. This redundancy not only
leads to no phenotype, but also makes the experimental interpretation difficult. What is
the solution to overcome the redundancy? With single-cell sequencing, it is feasible now to
profile all the enriched ion regulators given a cell type [
279
,
280
]. For example, abundant ion
regulators in different cell types within zebrafish somites and fins can be identified using this
technique. Alternatively, whole mount in situ hybridization can be performed systematically,
to examine their expression during zebrafish embryogenesis, as was performed in the case
of the potassium channel gene subfamilies [281,282]. Once their gene expression is known,
cell- and/or tissue-specific multiplexing gene knockout or knockdown by CRISPR can be
used to examine certain ion regulators’ function in developmental patterning [
283
–
287
].
Moreover, chemogenetic and optogenetic tools are also expected to be effective to override
endogenous ion regulators in a treated time window (Figure 3).
Cells 2023, 12, x FOR PEER REVIEW 19 of 33
Figure 3. Summary and perspectives of bioelectricity in zebrash developmental paerning re-
search. Bioelectricity may function in zebrash early embryos for paerning adult tissue/organs.
Multiple ion regulators exist in each cell, so the cell’s bioelectric properties are generally robust.
Minor disruptions (smaller lightning arrows) are not enough to change the bioelectric status quo,
and only signicant changes (big lightning arrows) may break through the robustness. This may
explain why most zebrash mutants were found with GOF (gain-of-function), DN (dominant neg-
ative), and ectopic expressions. Mechanistically, this bioelectric paerning may interact with al-
ready-known morphogen proteins and transcription factors. However, most likely, there are un-
known mechanisms that mediate this bioelectric paerning. Next-generation sequencing technolo-
gies and CRISPR genome editing may help decipher such novel mechanisms. In addition, the re-
cently developed genetically encoded tools for neuroscience, such as GEVIs, optogenetics, and
chemogenetics, are readily adopted to the embryonic paerning research eld. These tools allow us
to monitor and manipulate bioelectricity in a non-invasive manner.
5.3. Developmental Paerning by Bioelectric Memory
Another fundamental question about bioelectricity is how it works during embryo-
genesis. From the current data of the zebrash pigment mutants, cell–cell interactions are
critical for adult pigment paerning. Moreover, the interaction between the pigment cells
and other skin cells could also be important, as suggested by the transplant experiment of
the mau sh [110]. One key phenomenon of bioelectric paerning we have learned from
the Dhi2059 long-nned mutant is that the adult n paerning can be determined much
earlier, even back to the 1–2 dpf sh embryo stages [112]. The zebrash larva n buds have
not yet developed at this embryonic stage. Very likely, the transient alteration of bioelec-
tric properties of the two waves of n progenitor cells, which migrate from somites into
Figure 3.
Summary and perspectives of bioelectricity in zebrafish developmental patterning research.
Bioelectricity may function in zebrafish early embryos for patterning adult tissue/organs. Multiple
ion regulators exist in each cell, so the cell’s bioelectric properties are generally robust. Minor
disruptions (smaller lightning arrows) are not enough to change the bioelectric status quo, and only
significant changes (big lightning arrows) may break through the robustness. This may explain
why most zebrafish mutants were found with GOF (gain-of-function), DN (dominant negative), and
ectopic expressions. Mechanistically, this bioelectric patterning may interact with already-known
morphogen proteins and transcription factors. However, most likely, there are unknown mechanisms
that mediate this bioelectric patterning. Next-generation sequencing technologies and CRISPR
genome editing may help decipher such novel mechanisms. In addition, the recently developed
genetically encoded tools for neuroscience, such as GEVIs, optogenetics, and chemogenetics, are
readily adopted to the embryonic patterning research field. These tools allow us to monitor and
manipulate bioelectricity in a non-invasive manner.
Cells 2023,12, 1148 19 of 31
5.3. Developmental Patterning by Bioelectric Memory
Another fundamental question about bioelectricity is how it works during embryoge-
nesis. From the current data of the zebrafish pigment mutants, cell–cell interactions are
critical for adult pigment patterning. Moreover, the interaction between the pigment cells
and other skin cells could also be important, as suggested by the transplant experiment of
the mau fish [
110
]. One key phenomenon of bioelectric patterning we have learned from
the Dhi2059 long-finned mutant is that the adult fin patterning can be determined much
earlier, even back to the 1–2 dpf fish embryo stages [
112
]. The zebrafish larva fin buds
have not yet developed at this embryonic stage. Very likely, the transient alteration of
bioelectric properties of the two waves of fin progenitor cells, which migrate from somites
into the fin buds [
288
,
289
], is enough to change adult zebrafish fin size. This memory
of bioelectric property change may influence the differentiated cell interaction later in
the fin anlagens. This bioelectric memory concept has been proposed and validated in
the head–tail body axis determination in flatworms by the Levin research group [
290
].
Depolarizing edema cells during the first three hours after amputation is enough to cause a
double-headed phenotype in flatworms, indicating that bioelectric memory is critical for
patterning [
291
]. Thus, it is also reasonable that the bioelectric memory could be affected
in other reported long-finned mutants [
105
,
107
,
108
]. Based on this, we have proposed
a two-stage model for fin-size regulation [
112
]: the bioelectric memory may be formed
at the somite stage, before the fin progenitor cells migrate to the fin buds. Furthermore,
this memory can guide the local cell interactions and eventually determine the fin shape
and size. However, the bioelectric memory can also make the developmental patterning
mechanism more elusive. Generally, there will be a time lag for the phenotype develop-
ment after bioelectric perturbation. Moreover, the bioelectric memory formation might
occur within a short time window. Therefore, careful experimental design, including cell
lineage tracking, bioelectricity measurement, and perturbation, is essential to decipher this
intriguing mechanism.
5.4. Biological Pathways Downstream of Bioelectricity in Different Systems
Discovering unknown downstream signaling pathways of Vm on different functions
could help explain all the diverse functions of bioelectricity. There is still a long way before
we know how this biophysical cue is integrated into current biochemistry theory. As neural
action potentials can deliver a signal to a long-range destination along the axon and relay
by neurotransmitters, the non-excitatory embryonic tissues may use a similar mechanism
in an atypical way for guiding cell–cell interactions. Instead, the embryonic cells may
form weak bioelectrical networks [
8
]. Different cells and tissues may dominantly utilize
specific pathways.
The morphogen protein gradient is one of the most influential developmental pat-
terning principles with solid experimental support from many organisms, including ze-
brafish [
292
,
293
]. Relationships between bioelectricity and morphogen proteins, and their
downstream transcription factors, are particularly interesting (Figure 3). This information
will help us better understand how bioelectricity works and its interaction or cross-talks
with current already-known morphogen proteins. No clear experimental evidence was
reported in zebrafish yet, but multiple morphogen-signaling pathways were directly or
indirectly linked with bioelectricity in other organisms. For example, cell membrane rest-
ing potential can alter a frog’s brain development through Notch signaling [
294
]. BMP
signaling also mediates morphological changes of Kcnj2/Kir2.1 gene mutations in both
flies and mice [
295
–
297
]. However, the ion channel and WNT signaling relationship are
less clear in developmental biology, and there is limited evidence from pathological condi-
tions [
298
]. The most apparent case is bioelectricity and the hedgehog-signaling pathway.
Two transient receptor potential (TRP) channels, PKD1L1 and PKD2L1, modulate ciliary
calcium concentration, and the loss of these two channels leads to increased Gli1 activity
and subsequent hedgehog signaling [
299
,
300
]. Additionally, direct evidence was reported
that optogenetic depolarization was found to promote smoothened membrane localization
Cells 2023,12, 1148 20 of 31
and increase hedgehog signaling, which also promotes cellular depolarization in the fly
wing disc [
301
]. Furthermore, two critical components of this pathway, DISP1 and PTCH1,
function as cation-powered transporters. DISP1 utilizes the transmembrane sodium ion
gradient to release cholesterylated SHH from HEK293 cells. In comparison, the PTCH1
receptor behaves as a K
+
-powered cholesterol transporter, which employs a transmembrane
potassium ion gradient to antagonize SMO with cholesterol in NIH-3T3 cells [
302
]. Another
report found the PTCH1 inhibits SMO and depends on extracellular sodium ion concentra-
tion [
303
]. Thus, bioelectricity may modulate multiple steps of hedgehog signaling from
SHH secretion (establishment of morphogen gradients) to SHH reception (interpretation of
morphogen), indicating bioelectricity patterning may be mediated by morphogen-signaling
pathways, at least partially.
6. Conclusions
Bioelectricity has emerged as a new player in developmental patterning and organ
size control. Here, we focused on the zebrafish model system and systematically reviewed
genetic evidence of bioelectricity from zebrafish mutants. Additionally, we briefly sum-
marized the newly developed genetically encoded voltage indicators and cellular voltage
manipulators (optogenetics and chemogenetics) and their potential to be used for zebrafish
bioelectricity research. Finally, we discussed future directions and opportunities for bio-
electricity research in developmental patterning.
Author Contributions:
Conceptualization, G.Z.; writing—original draft preparation, M.R.S. and
G.Z.; writing—review and editing, M.R.S. and G.Z. All authors have read and agreed to the published
version of the manuscript.
Funding:
This research was supported by the National Institute of General Medical Sciences of the
National Institutes of Health (2R35GM124913) to G.Z. The content is solely the responsibility of the
authors and does not necessarily represent the official views of the funding agents.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
We thank Zhang lab members Sung Jun Park, Ziyu Dong, and Dingxun Wang
for proofreading this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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