Download Development of zebrafish epidermis

Birth Defects Research (Part C) 93:205–214 (2011)
REVIEW
Development of Zebrafish Epidermis
Wei-Jen Chang and Pung-Pung Hwang*
Zebrafish epidermal ionocytes are analogous to mammalian kidney cells
in terms of expression and function of ion transporters. In this review,
we summarize current findings about the development of the zebrafish
epidermis and demonstrate how the zebrafish regulate stress acclimation through induction of cell differentiation. In addition, cellular homologies between zebrafish epidermal ionocytes and mammalian kidney
cells are presented to show the potential of zebrafish epidermis as an in
vivo model to study the development and function of mammalian
cells. Birth Defects Research (Part C) 93:205–214, 2011. VC 2011
Wiley-Liss, Inc.
Key words: zebrafish; ionocyte; epidermis development
INTRODUCTION
The epidermis is the biggest organ
and the first barrier of vertebrates,
and epidermal cells are specified
very early during embryological
development. However, epidermal
cell specification and terminal differentiation
during
vertebrate
ontogenesis is not completely
understood.
Teleost
epidermis
exhibits more complex cellular
composition than amniote epidermis; teleost epidermis protects the
organism from sterner aquatic
environments. In addition to keratinized cells, teleost epidermis
develops unicellular glands and
ion-regulated
cells
that
are,
respectively, similar to mammalian intestine cells and kidney
cells. However, the mechanism of
specification of these epidermal
cell types in teleosts remains
unclear. In this review, we
describe the structure and cellular
composition and summarize cur-
rent findings on the development
of epidermis and the differentiation of epidermal cells. We also
discuss how epidermal cell differentiation is involved in the functional regulation of fish skin during
acclimation to a sterner and fluctuating environment and propose
zebrafish epidermis as an appropriate in vivo model for the physiological and developmental studies
of mammalian kidney based on
their functional and developmental
homologies.
STRUCTURE OF TELEOST
SKIN EPIDERMIS
In amniotes or aquatic vertebrates, one of the most important
functions of skin is providing effective barriers between the organism and its environment, and the
skin comprises physical, chemical/
biological (antimicrobial, innate
immunity), and adaptive immuno-
logical barriers (Hawkes, 1974;
Proksch et al., 2008). Fish skin,
similar to mammalian skin, is
composed of three compartments,
the epidermis, dermis, and hypodermis; however, fish are not
identical to mammals in the cellular components and structure of
epidermis. One of the most
obvious difference is that the teleost epidermis has only living cells
within it. Unlike the terrestrial vertebrate’s epidermis, which is covered by an outer layer of keratinized dead cells, teleost skin surface is composed of living cells
that are covered with mucus but
not cornified envelope (Hawkes,
1974; Concha et al., 2003; Le
Guellec et al., 2004). Another difference is that mammalian epidermis is a well-organized stratified
tissue with basal, spinous, granular, and horny cells from the basal
membrane to the skin surface,
while teleost epidermis does not
have this kind of cell order and is
composed of various cell types
(Henrikson and Matoltsy, 1967a;
Alonso and Fuchs, 2003). These
morphological differences in epidermis may be ascribed to the different environments inhabited by
terrestrial and aquatic animals.
Teleost epidermis can be subdivided into surface, intermediate,
and basal layers (Fig. 1). The surface layer is a single cell layer that
develops microridges at the outer
surface. Even though these cells
Supported by grants from the National Science Council and Academia Sinica of Taiwan, ROC (to P.P.H. and W.J.C.)
Wei-Jen Chang is from Molecular and Biological Agricultural Science Program, Taiwan International Graduate Program, National
Chung-Hsing University and Academia Sinica, Taipei, Taiwan; Institute of Cellular and Organismic Biology, Academia Sinica, Taipei,
Taiwan; and Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan
Pung-Pung Hwang is from Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan
*Correspondence to: P. P. Hwang, Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei 115, Taiwan.
E-mail: pphwang@gate.sinica.edu.tw
View this article online at wileyonlinelibrary.com. DOI: 10.1002/bdrc.20215
C 2011 Wiley-Liss, Inc.
V
206 CHANG AND HWANG
Figure 1. Upper panel displays the structure of embryonic epidermis, which consists of outer EVL and inner EBL. Lower panel demonstrates the structure of adult epidermis, which consists of surface layer, intermediate layer, and basal layer. Surface layer and
basal layer are single cell layers that is composed of keratinocytes (K) and undifferentiated cells, respectively. Intermediate layer is
composed of mucous cells (M), club cells (C), ionocytes (I), and undifferentiated cells.
are rich in keratin filaments, they
do not produce stratum corneum,
as horny cells do in other vertebrates. Moreover, cells within this
epidermal layer are not periodically
renewed, but they are replaced
individually on cell death (Le Guellec et al., 2004). The intermediate
layer is composed of various types
of cells, including unicellular glands
(such as mucous cells and club
cells), sensory cells, ionocytes, and
the most dominant undifferentiated cells. Each of these cell types
has specific function: mucous cells
secret a mucus coat that covers
the epidermis and protects fish
from infection of bacteria, fungi,
and parasites; club cells release
alarm signal factors to their surroundings and exhibit a fright reaction via olfactory stimulation; ionocytes, which present ion transporters at the plasma membrane,
maintain the homeostasis of body
fluids; the undifferentiated cells
can divide rapidly and replace dead
surface cells or intermediate cells
in the epidermis (Henrikson and
Matoltsy, 1967a, 1967b, 1967c;
Concha et al., 2003; Le Guellec
et al., 2004). The basal part of the
epidermis is a single cell layer (basal layer), which is attached to the
basement membrane via hemidesmosomes. The basal layer tightly
links epidermis to dermis. In addition, during skin development, the
basal layer is associated with the
production of early collagenous
stroma and the development of
skeletal elements in dermis. In
addition, the basal layer is suggested to affect the process of epidermal substance deposition at the
posterior surface of the scales
(Sire and Huysseune, 2003; Le
Guellec et al., 2004).
Aquatic animals face harsh and
fluctuating environments; hence,
their epidermis functions to protect them from fluctuating temperatures, osmotic stress, microorganism infection, and frequent
physical strikes. To cope with
these environmental stresses, tel-
Birth Defects Research (Part C) 93:205–214, (2011)
eost fish have developed various
types of epidermal cells that carry
out distinct functions.
DEVELOPMENT OF TELEOST
SKIN EPITHELIUM
The epidermis of zebrafish embryo
consists of two cell layers: the surface layer or enveloping layer
(EVL). EVL has morphological and
functional similarities to the periderm of mammalian embryo. The
inner layer is called the epidermal
basal layer (EBL) (Fig. 1). Different from the basal layer of adult
epidermis, EBL contains both differentiated and undifferentiated
cells. These two cell layers are
formed at different developmental
stages and become the simple epithelium of the embryo (M’Boneko
and Merker, 1988; Kimmel et al.,
1990). EVL is generated during
the blastula period, and within this
developmental stage the blastoderm is specified into three cell
populations: the surface blasto-
DEVELOPMENT OF ZEBRAFISH EPIDERMIS 207
derm becomes EVL; the vegetal
cells that attached and fused with
yolk cells become the yolk syncytial layer (YSL); and the cells
between EVL and YSL are the deep
cells. EVL not only functions in
protecting the embryo as the
mammalian periderm but also
tightly joins YSL and the deep
cells. EVL is essential to blastoderm morphogenesis, as EVL cells
drag the deep cells along with YSL
cells (Kimmel et al., 1990; Slanchev et al., 2009). On the other
hand, the EBL starts to form at the
gastrula period after formation of
the three embryonic layers. During
gastrulation stage, the three germ
layers, ectoderm, mesoderm, and,
endoderm, are formed and cell
specification is turned on (Heisenberg and Tada, 2002). Epidermal
ectoderm (the EBL), preplacodal
ectoderm, neural crest, and neural
plate arise from ectoderm, and the
specification of these four cell patterns starts after the determination
of dorsal–ventral axis. The EBL
covers the whole embryonic surface at the end of gastrulation, and
the formation of the two-layered
(including EVL and EBL) simple epithelium completes the process
(Cherdantseva and Cherdantsev,
2006; Little and Mullins, 2006).
The simple epithelium becomes
further stratified and develops the
three-layered structure as previously described during metamorphosis (Le Guellec et al., 2004).
EVL is the first specified cell type
that arises at mid-blastula stage
during embryonic development,
and the adhesion or proximity
between EVL cells and the neighboring cells is essential for EVL
specification (Sagerstrom et al.,
2005). In recent years, several
maternal
factors
have
been
reported to affect EVL development, and all of these genes are
related to epiboly progression.
Two transcription factors, IRF6
and Pou5f1 (Oct4), are necessary
for the expression of EVL-specific
genes and negatively regulate
lamellipodia formation of EVL cells,
respectively (Lachnit et al., 2008;
Sabel et al., 2009). A Smad2
cotranscription factor, FoxH1, is
associated with the keratin genes
expression in EVL (Pei et al.,
2007). A cell adhesion molecule,
epithelial cell adhesion molecule
(EpCAM), is required for EVL cell
protrusive and migrant abilities
(Slanchev et al., 2009). A kinase
of IjB, IKK1, is essential for EVL
differentiation (Fukazawa et al.,
2010). However, the connections
between these factors in regulation of EVL differentiation are still
uncertain.
Regulation of EBL development
is better understood, and its pathways are more conserved between
fish and mammals than those of
EVL. Specification of basal layer
cells, which is regulated by bone
morphogenetic protein (BMP) signaling, accompanies dorsal–ventral axis formation during zebrafish gastrulation (Yamamoto and
Oelgeschlager, 2004; Little and
Mullins, 2006). Differential expression of BMP and BMP antagonists
turn on specific downstream
genes, and thus cause the development of epidermal ectoderm,
preplacodal
ectoderm,
neural
crest, and neural plate. During
late
blastula
stage,
dorsally
expressed
BMP
induces
the
expression of transcription factors
critical for nonneural ectoderm
specification,
including
foxi1,
gata3, tfap2a, tfap2c, and DNp63.
Knockdown experiments showed
that foxi1, gata3, tfap2a, and
tfap2c are required for specification of preplacodal ectoderm
(Kwon et al., 2010), whereas
DNp63 is essential for the development of epidermal ectoderm (Lee
and Kimelman, 2002). DNp63 is
the isoform of transcription factor
p63, a member of p53 family, produced by an alternative promoter
(Aberdam et al., 2007). DNp63
lacks the N-terminal transactivation domain and the DNA binding
domain, which is highly conserved
in the p53 family and is considered
a dominant-negative regulator of
p53 and p63 (Lee and Kimelman,
2002; Yamamoto and Oelgeschlager, 2004). Moreover, DNp63, a
direct target of BMP signaling, is
specifically expressed in nonneural
ectoderm and it not only induces
the specification of epidermal
ectoderm but also inhibits the
neural ectoderm formation during
the early gastrulation (Bakkers
et al., 2002; Aberdam et al.,
2007). After gastrulation, DNp63
becomes the marker of proliferating epithelial cells, and it is
required for cell proliferation of
epidermis; however, expression of
mature epidermal genes (e.g.,
keratin) and differentiation of epidermal cells was not blocked in
DNp63 morphants (Lee and Kimelman, 2002).
Taken together, the epidermis of
zebrafish embryo is composed of
EVL and EBL. The mechanisms responsible for the regulation of EVL
formation are still unclear, and the
specification of EBL is directed by
BMP-induced DNp63. Thereafter,
EBL is specified into different subtypes of epidermal cells, but the
regulation of specification of these
cells has not been established.
SPECIFICATION AND
DIFFERENTIATION OF
EPIDERMAL CELLS
Different types of teleost epidermal cells were identified more
than 40 years ago (Henrikson and
Matoltsy, 1967a, 1967b, 1967c),
while the specification and differentiation of these cells are largely
unknown to date. Here, we summarize recent findings on epidermal cell differentiation, especially
focusing on ionocytes which are
well-studied so far.
Hsiao et al. (2007) first proposed
a regulatory pathway for ionocyte
differentiation and specification in
zebrafish, and this model was
mostly supported and further
explored in subsequent studies
(Fig. 2) (Janicke et al., 2007; Esaki
et al., 2009; Hwang and Perry,
2010; Hwang et al., 2011). Janicke
et al. (2007) proved that ionocytes
are derived from nonneural ectoderm via lineage tracing experiments. Further, ionocyte progenitor
markers are transiently expressed
in DNp63-positive undifferentiated
cells during somitogenesis, but
knocking-down DNp63 expression
showed no effects on proliferation
and differentiation of ionocytes
(Hsiao et al., 2007; Janicke et al.,
Birth Defects Research (Part C) 93:205–214, (2011)
208 CHANG AND HWANG
Figure 2. Specification pathways of zebrafish epidermal cells. Cell markers are presented according to expression time, and regulatory factors are shown to indicate the
effecting point. Notch signaling determines the cell lineages of ionocyte and keratinocyte. Foxi3a promotes the differentiation of ionocyte, and Gcm2 is required to specify
HR cells. Differential expression of Foxi3a/3b and series expression of Jagged family
are proposed to control the specification of ionocyte subtypes. Dotted arrows indicate
the possible pathways that are still uncertain.
2007). Therefore, ionocytes and
keratinocytes are derived from the
same progenitor cells that express
DNp63, but the development of ionocytes is independent of DNp63.
The cell specification markers of
ionocytes are first presented in
ventral epidermis after gastrulation (bud stage), including a Forkhead-box I transcription factor,
foxi3a, and a ligand of Notch signaling, deltaC (Hsiao et al., 2007).
Later on, foxi3b (homolog of
foxi3a) and other Notch ligands,
jagged1a and jagged1b, are
expressed in epidermal cells at 5somite stage, while jagged2a and
jagged2b show similar expression
patterns at the 11-somite stage
(Hsiao et al., 2007; Janicke et al.,
2007). The analysis of mutants,
loss-of-function, gain-of-function,
transgenic zebrafish, and other
molecular/cellular
approaches
have shown that Delta-Notchmediated lateral inhibition determines cell lineages of ionocytes
and keratinocytes, and Foxi3a was
proven to be essential for ionocyte
differentiation (Hsiao et al., 2007;
Janicke et al., 2007). Based on the
findings using these two master
regulators, the Delta-Notch signaling and the Forkhead-box tran-
Birth Defects Research (Part C) 93:205–214, (2011)
scription factor class I, a regulatory
model of ionocyte differentiation
was proposed. After gastrulation,
subtypes
of
epidermal
cells
(including ionocytes and keratinocytes) start to be specified and a
subset of DNp63-positive undifferentiated cells turns on the expression of deltaC and foxi3a. DeltaC
further inhibits expressions of foxi3
and other ionocyte markers in
neighboring cells through Notch
signaling, and Notch signaling
maintains the cell fate of keratinocyte lineage. On the other hand,
DNp63 is downregulated in deltaC/
foxi3a-expressing cells, and thereafter Foxi3a triggers cell differentiation and turns on the expression
of mature ionocyte markers.
The mature ionocytes contain
different subtypes whose lineages
result from differentiation during
embryonic development. Four subtypes of ionocytes have been identified to date: (1) Na1-K1-ATPase
rich (NaR) cells, (2) H1-ATPaserich (HR) cells, (3) Na1-Cl2
cotransporter (NCC) expressing
cells, and (4) K1-secreting (KS)
cells (Lin et al., 2006; Wang et al.,
2009; Abbas et al., 2011; Hwang
et al., 2011). However, the regulatory mechanisms that control
the specification of ionocyte subtypes are not clear. Previous studies provided two possible mechanisms that may govern this cell
fate determination. One is that
Foxi3a turns on the expression of
foxi3b at the 5-somite stage, and
then Foxi3a and Foxi3b are downregulated in NaR cells and HR
cells, respectively (Hsiao et al.,
2007; Esaki et al., 2009). This differential expression of Foxi3a and
Foxi3b may be critical for the
specification of ionocyte subtypes.
Another is that the expression of
jagged1a and jagged1b was
detected at 5-somite stage, and
Foxi3a was reported to trigger
the expression of jagged2a and
jagged2b at the 11-somite stage
(Janicke et al., 2007). These
results indicate that Notch signaling may also participate in specification of ionocyte subtypes when
triggered by different ligands.
Besides Foxi3 and Notch signaling, we have demonstrated that
DEVELOPMENT OF ZEBRAFISH EPIDERMIS 209
another transcription factor controls the specification of HR cells
(Chang et al., 2009). A cell faterelated transcription factor, glial
cell missing 2, gcm2, is expressed
from bud stage in the zebrafish
ectoderm, and its mRNA is specifically localized in HR cells. Gainand loss-of-function experiments
showed that Gcm2 is essential for
differentiation of HR cells but not
NaR cells (Chang et al., 2009). A
subsequent study by Esaki et al.
(2009) further indicated that
knockdown of Gcm2 suppressed
Foxi3a expression at 24 hpf. This
implies that Gcm2 may affect the
differential expression of Foxi3a/
Foxi3b in ionocyte progenitors and
determine the NaR cell and HR cell
lineages. In addition to another
member of foxi family, foxi1, was
shown to be required for HR cell
differentiation (Esaki et al., 2009).
However, how Foxi1 regulates HR
cell differentiation is not clear
because expression of deltaC and
foxi3a were blocked in foxi1 morphants, but only HR cells (not NaR
cells) failed to be differentiated in
foxi1 mutants (Esaki et al., 2009).
Furthermore,
foxi1
transcripts
were detected in nonneural ectoderm during gastrulation but
remained undetected in HR cells
or ionocyte precursors during
somitogenesis. These results suggest that Foxi1 may function in HR
cell differentiation through indirect
regulation.
The specification of ionocytes
begins after gastrulation, and
gcm2 starts to be expressed at the
bud stage. Thereafter, Foxi3a
induces foxi3b and jagged2a/2b
expressions at the 5-somite and
11-somite stages, respectively,
and the expression of atp1b1b
(Na1-K1-ATPase b subunit, a
mature marker of ionocytes) is
also turned on at the 11-somite
stage. At the 14-somite stage,
DeltaC and DNp63 are downregulated in the ionocyte lineage, and
shortly after (at 15-somite stage),
atp6v1a (H1-ATPase subunit A, a
mature marker of HR cells) is
present in epidermis. Na1-K1ATPase and H1-ATPase can be
weakly detected at 24 hpf, and
the differentiation of ionocytes is
considered to be completed (Hsiao
et al., 2007; Janicke et al., 2007).
Until now, information concerning
the detailed pathways of ionocyte
subtype specification is still scarce
and fragmentary. It is suggested
that differential expression of
Foxi3a/Foxi3b and/or Notch signaling may specify the ionocyte
subtypes. Moreover, the differentiation of HR cells depends on
Gcm2, and may be mediated by
Foxi1 in some way; however, the
detailed mechanisms need to be
further studied (Chang et al.,
2009; Esaki et al., 2009).
In contrast to ionocytes, the differentiation pathways of other cell
types in epidermis are largely
unknown. DNp63 is required for
the proliferation of keratinocyte
precursors but is not involved in
keratinocyte differentiation (Lee
and Kimelman, 2002). Some studies related to zebrafish keratinocyte differentiation have been
reported in recent years. For
instance, a keratinocyte differentiation defect was observed in the
mutant strain, psoriasis, which
may encode a secret factor (Webb
et al., 2008). Ectopic expression of
human paired-like homeodomain
transcription factor 2c, which is
required for differentiation of
human keratinocytes, caused a
thickening of the epidermis, formation of horn-like structure, and
increased level of Keratin 8 expression in zebrafish embryos (Shi
et al., 2010). However, the endogenous regulation of keratinocyte
differentiation is still unclear. On
the other hand, a member of
Grainyhead/CP2 transcription factor family, grhl1, was identified as
a cell marker of ‘‘non-keratinocyte’’ lineage (including ionocytes
and pvalb8-expressing cells) during specification of epidermal cells,
but grhl1 is dispensable for the differentiation of all the investigated
epidermal cell types. Moreover, the
pvalb8-expressing cells, putative
mucous cells, were increased in
foxi3a morphants, suggesting that
pvalb8-expressing cells share the
same precursor pool with ionocytes
(Janicke et al., 2010).
Teleost
epidermis
contains
diverse cell types, and each of
them has distinct functions for
protection and acclimation. Specification and differentiation of these
cell types are still budding fields,
and more efforts are needed.
CELL DIFFERENTIATION
AND ENVIRONMENTAL
ACCLIMATION
Epidermis is like an adjustable
armor of teleosts; it not only provides physical, chemical, and biological protection but also is able to
rearrange its composition for environmental acclimation. Analysis of
the incorporation of tritiated thymidine (3H-thymidine) has revealed
that cells within the intermediate
and basal layers of epidermis have
strong cell proliferation potential in
adult teleost (Genten et al., 2009).
This result was confirmed by 5bromo-20 -deoxyuridine incorporation and DNp63 staining, and it was
shown that proliferating epidermal
cells were restricted in the intermediate and basal layers (Chen et al.,
2011). The basal layer cells of teleost epidermis displayed the ability
to differentiate rapidly during
wound healing (Quilhac and Sire,
1999). In addition, cell proliferation
and differentiation of epidermal
ionocytes were observed in zebrafish embryos during acclimation of
acid stress. Based on immunostaining, mature ionocyte, DNp63expressing cells, and proliferating
cell nuclear antigen (PCNA)-positive cells all increased after acid
acclimation, and terminal deoxynucleotidyl transferase dUTP nick end
labeling
(TUNEL)
assay
also
showed that cell apoptosis did not
decrease in the acid-treated group.
These results demonstrate that
environmental stress can induce
proliferation of undifferentiated epidermal cell and promote differentiation of ionocytes, and this cell increment benefits zebrafish during
stress acclimation (Horng et al.,
2009).
Cell proliferation and differentiation had been observed in gill epithelium, and since 1960s it has
been thought that the rearrangement of cell composition improves
functional regulation for stress
Birth Defects Research (Part C) 93:205–214, (2011)
210 CHANG AND HWANG
response. Based on autoradio3
graphic
analysis
H-thymidine
incorporation, cells involved in
regeneration of gill epithelium were
found in gill filament, and gill epithelium cells exhibited greater turnover rates when the teleost were
transferred from fresh water to sea
water (Conte and Lin, 1967). Similarly, 5-bromo-20 -deoxyuridine-labeled gill epithelium cells also
showed higher turnover rates in
seawater, and it further showed
that ionocytes within gill epithelium
are replaced continuously by newly
differentiated cells (Uchida and
Kaneko, 1996). In addition to
osmoregulation, cell proliferation of
gill epithelium was proven to regulate different stresses. After exposing the tilapia (Oreochromis mossambicus) to a high copper environment, both PCNA-positive cells and
ionocytes that undergo cell death
increased, revealing that exposure
to copper promotes cell turnover of
gill epithelium (Dang et al., 2000).
Although cell differentiation was
considered to regulate ionocyte
function for years, nothing is known
about the molecular mechanism
within the regulatory process. Studies in ionocyte differentiation provide a powerful tool to ask detailed
physiological questions. One example is the cellular regulation in
zebrafish during acid acclimation.
The duplication of HR cells was
observed in both embryo epidermis
and adult gill epithelium after acclimation of zebrafish in acidic environment (Chang et al., 2009; Horng
et al., 2009), and the induction of
H1-ATPase and stimulation of
gcm2, essential for HR cell differentiation, were detected during acid
acclimation (Chang et al., 2009).
These results suggest that under
acid stress, zebrafish turn on the
expression of gcm2 and the differentiation of HR cells is promoted to
maintain the acid–base homeostasis. On the other hand, cold-acclimated zebrafish possess more ionocytes in gill epithelium, and this is
regulated through decreasing cell
turnover rate by downregulation of
cell division/apoptosis and upregulation of cell differentiation (Chou
et al., 2008). Differentiation of embryonic and adult ionocytes may be
regulated by the same or similar
mechanisms.
In addition to intracellular regulation, upstream factors (i.e., neuroendocrine factors) are known to
promote
osmoregulation
via
inducing differentiation of different
ionocytes. It is suggested that
growth hormone induces sea
water-type ionocyte formation,
prolactin induces fresh water ionocyte formation, and cortisol interacts with these two hormones to
regulate
cell
differentiation
(McCormick,
2001;
Sakamoto
et al., 2001). In zebrafish, the
transcription level of atrial natriuretic peptide, rennin, prolactin,
growth hormone, and parathyroid
hormone were found to respond to
salinity changes (Hirose and Hoshijima, 2007). However, it remains
unknown as to how these endocrine factors regulate ionocyte differentiation. Based on the knowledge of ionocyte development,
isotocin, a homolog of oxytocin,
was found to regulate proliferation
of epidermal progenitors and differentiation of ionocytes. Isotocin
transcripts were increased in
zebrafish embryo after hypoosmotic shock. Gain- and loss-offunction experiments show that
isotocin not only enhances the
expression of foxi3a and foxi3b
but also increases the numbers of
epidermal progenitors and ionocytes (Chou et al., 2010).
The studies of epidermal cell differentiation have enabled us to
ask detailed physiological questions about environmental acclimation of teleosts. For instance, a
neuroendocrine
stress-response
(such as isotocin) may turn on the
differentiation process of ionocytes
and further regulate the homeostasis of teleosts to acclimate to
environmental stress.
ZEBRAFISH EPIDERMIS
AS AN IN VIVO MODEL FOR
MAMMALIAN
PHYSIOLOGICAL AND
DEVELOPMENTAL STUDIES
Zebrafish has emerged as a
potential vertebrate model in various research fields. Based on their
Birth Defects Research (Part C) 93:205–214, (2011)
characteristics, such as high fecundity, short generation time,
external development, and transparent embryo, zebrafish is suitable for gene manipulation and observation. Provided with the techniques that are essential for
genetic analysis, mutagenesis and
large scale screens are well-established in zebrafish. Having these
advantages, zebrafish have been a
competent animal model for studies on development, physiology,
human disease, and drug discovery (Dooley and Zon, 2000;
Lieschke and Currie, 2007; Liu and
Leach, 2011).
The structure and cellular composition of teleost epidermis is different from mammalian epidermis,
and specifically, the intermediate
layer of teleost epidermis contains
various mature cells, such as ionocytes and mucous cells. Although
these cells do not exist in the
mammalian epidermis, they are
structurally similar to some of the
mesoderm- or endoderm-derived
cells of mammals. Mucous cells
present the same characteristics as
the goblet cells in the mammalian
intestine (Shih et al., 2007),
whereas ionocytes exhibit similar
structure and function to mammalian kidney cells that are responsible for ionic reabsorption and acid–
base regulation (Hwang, 2009;
Hwang and Perry, 2010). Different
segments of mammalian kidney
possess a variety of ion-transporting cells that are involved in distinct functions of ionoregulation/
osmoregulation and acid–base regulation. Analogous to mammalian
kidney cells, teleost ionocytes also
have different subtypes that carry
out respective transport functions
(Fig. 3). NaR cells absorb Ca21
through a pathway similar to that
for Ca21 reabsorption in the mammalian kidney. Extracellular Ca21 is
transported via the epithelium
Ca21 channel that is expressed at
the apical membrane, and then
Ca21 is extruded into interstitial
fluid through the basolateral Na1/
Ca21 exchanger (NCX) and plasma
membrane Ca21-ATPase (PMCA)
(Hoenderop et al., 2000; Pan
et al., 2005; van de Graaf et al.,
2006; Liao et al., 2007). HR cells
DEVELOPMENT OF ZEBRAFISH EPIDERMIS 211
Figure 3. Models of zebrafish ionocytes and mammalian kidney cells. Details refer to the text (Section Zebrafish epidermis as an in
vivo model for mammalian physiological and developmental studies). Ionocytes: Na1-K1-ATPase rich (NaR) cell, H1-ATPase-rich
(HR) cell, Na1-Cl2 cotransporter (NCC) cell, and K1-secreting (KS) cell. Kidney cells: distal convoluted tubule (DCT) cell, a-intercalated cell (a-IC), proximal tubular (PT) cell, and thick ascending limb (TAL) cell. AE1, anion exchanger 1b; HA, H1-ATPase; NHE,
Na1/H1 exchanger; Rhcg, Rhesus glycoprotein; NKA, Na1-K1-ATPase; ECaC, epithelial Ca21 channel; PMCA, plasma membrane
Ca21-ATPase; NCX, Na1/Ca21 exchanger; NCC, Na1-Cl2 cotransporter; Kcnj1/ROMK, potassium channel.
are characterized by expression of
H1-ATPase at the apical membrane, and coupled with basolateral anion exchanger 1 (AE1), regulate acid–base balance via excretion of H1 and absorption of
bicarbonate. HR cells not only secret acid as do the a-intercalated
cells (ICs) in the mammalian collecting duct but also absorb Na1 by
an apical Na1/H1 exchanger (NHE)
like mammalian proximal tubular
cells. In addition, HR cells function
in ammonia excretion through an
apical Rhesus glycoprotein (Rhcg1)
(Lin et al., 2006; Purkerson and
Schwartz, 2007; Yan et al., 2007;
Shih et al., 2008; Horng et al.,
2009; Wagner et al., 2009; Lee
et al., 2011). NCC cells, similar to
mammalian
distal
convoluted
tubule cells, express a Na1-Cl2
cotransporter at the apical membrane and are responsible for Na1
and Cl2 absorption (Reilly and Ellison, 2000; Wang et al., 2009). KS
cells apically express a potassium
channel, Kcnj1, and produce K1
efflux as do the mammalian thick
ascending limb cells of Henle’s loop
(Hebert et al., 2005; Abbas et al.,
2011).
In addition to morphological and
functional similarity, ionocytes are
also analogous to mammalian kidney cells in terms of a cell differentiation pathway. The main subtypes of collecting duct ICs are aICs and b-ICs, and they regulate
acid–base homeostasis in mammals via active transport of H1
and bicarbonate (Wagner et al.,
2009). In the adult kidney, the
proportions of a-ICs and b-ICs
appear to shift under chronic pH
imbalances, and an extracellular
matrix protein, hensin, mediates
this cell transformation (Schwartz
and Al-Awqati, 2005; Gao et al.,
2010). This suggests that IC subtypes have differentiation plastic-
Birth Defects Research (Part C) 93:205–214, (2011)
212 CHANG AND HWANG
ity that is essential for regulation
of their functions. However, it is
difficult to trace IC specification
during embryonic development in
mammal kidney, thus the mechanisms underlying IC specification
are largely unknown. A mouse mutant that fails to produce transcription factor, Foxi1, showed the
defect in IC differentiation, demonstrating an important role for Foxi1
in establishing the IC lineage
(Blomqvist et al., 2004; Vidarsson
et al., 2009). In addition, gain- and
loss-of-function experiments indicated that Notch signaling is
required for the development of the
mammalian renal collecting duct
and determines cell lineages of
principal cells and ICs (Jeong et al.,
2009). Mammalian ICs and teleost
ionocytes are not only similar in cell
structure but also have similar differentiation processes that are
determined by Notch signaling and
promoted by Foxi transcription factors (Hsiao et al., 2007; Janicke
et al., 2007). Similar differentiation
processes were also observed in
epidermal cells of the African
clawed frog (Xenopus laevis). Quigley et al. (2011) identified two
types of X. laevis epidermal cells
that have a similar cellular structure to mammalian a-ICs and bICs, respectively, and they termed
the two cell types as a-intercalating
and b-intercalating nonciliated cells
(INCs). According to inducibleoverexpression experiments, the
cell lineage of INCs is regulated by
Notch signaling, and Foxi1 acts
downstream of Notch to activate
INC differentiation (Quigley et al.,
2011). These studies suggest that
the developmental process of ionregulatory cells is conserved among
different vertebrates. Gene manipulation in zebrafish is considerably
easier compared to mammalian
models, and ionocytes in zebrafish
epidermis are easily traced to analyze the differentiation pathways of
each subtypes. Taken together,
zebrafish ionocyte is proposed to
be a competent in vivo model to
study IC differentiation.
Acid-based homeostasis is critical for maintenance of normal cellular function and is achieved by aICs and b-ICs in the kidney. It was
suggested that b-ICs may convert
to a-ICs in response to acidosis
(Al-Awqati, 2011). In zebrafish,
cell differentiation of HR cells is
turned on in response to an acidic
environment (Chang et al., 2009;
Horng et al., 2009). Studies on
zebrafish ionocytes may provide
some cues for the transport physiology and functional regulation in
mammalian kidney. Moreover, the
distinct functions of each subtype
of ionocytes could be analyzed in
vivo by scanning ion-selective
electrode techniques (Lin et al.,
2006; Shih et al., 2008), further
indicating the applicability of
zebrafish epidermis as a model for
the studies on the transport physiology and development biology of
mammalian kidney.
Epidermis is the outmost cell
layer of zebrafish, and therefore is
excellent for morphological observation, pharmacological/chemical
treatments, and functional analyses. Molecular, cellular, pharmacological, and physiological experiments can be conducted in vivo in
zebrafish skin. Moreover, zebrafish
epidermal cells exhibit similar cell
structure, cell function, and developmental processes to those in
mammalian kidney cells, intestine
cells, and skin cells as discussed
above. Taken together, zebrafish
skin is a potential in vivo model
for studies on the physiology, development, and disease pathogenesis in mammalian kidney cells,
intestine goblet cells, and skin keratinocytes.
CONCLUSION
The work presented herein shows
how the model of cell differentiation of zebrafish epidermis may be
useful not only in vertebrate physiological studies but also in functional and developmental studies
of mammalian kidney. As the regulatory mechanisms of teleost epidermal cell differentiation are not
completely understood, additional
in-depth investigations on the differentiation model of zebrafish
epidermal cells should bring more
solutions to solve the problems of
stress response and cell differentiation in mammalian kidney.
Birth Defects Research (Part C) 93:205–214, (2011)
ACKNOWLEDGMENTS
We thank L.Y. Huang, Y.C. Tung,
and S.A. Cruz for their technical
and secretarial assistance.
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