Discussion
In this study, 10 differentially expressed genes related to skin feeding
of discus fish were selected, among which MRC, ITGA11 and PTPRJ were
up-regulated in P_S period, JUNB, DPP3, CASP3, PPID, ALOXE3, HBE and
GALE were down-regulated in P_S period. 9 differentially expressed
genes were selected in brain of discus fish, among which EDNRB, LAPTM,
FOXB, OTX2, NRIF2 and SOX1 were up-regulated in P_B period, HBE, PRKCD
and H1-5 were down-regulated in P_B period.
One of arachidonic acid, the precursor of eicosanoids as paracrine
hormones involving in various processes important in human health or
disease, includes prostaglandins (PGs), leukotrienes and thrombox-anes.
Arachidonic acid were the enriched fatty acid metabolisms (P. Krieg et
al., 2002; Yoo et al., 2018). Arachidonate lipoxygenase3 (ALOXE3) gene
is a member of the lipoxygenase family, which are catabolized by
arachidonic acid-derived compounds and they are members of the epidermal
subfamily of mammalian LOX with preferential expression in the skin and
several other epithelial tissue (Peter Krieg et al., 2013). In this
study, it was found that the expression of discus fish skin was
different in NP_S and P_S periods, and decreased in P_S periods
compared with NP_S periods. It encoded enzyme is a hydroperoxide
isomerase that synthesizes a unique type of epoxy alcohol
(8R-hydroxy-11R, 12R-epoxyeicosa-5Z, 9E, 14Z-trienoicacid) from
12R-hydroperoxyeicosatetraenoic acid (12R-HPETE). This epoxy alcohol can
activate the nuclear receptor peroxisome proliferator-activated receptor
alpha (PPARalpha), which is implicated in epidermal differentiation1
(Arias-Andres, Kettner, Miki, & Grossart, 2018; Yoo et al., 2018). Have
study reported demonstrate the epidermal-type lipoxygenase. Aloxe3 is a
potentially novel effector of the therapeutic fasting response (Sala et
al., 2018). The discus fish will damage the skin tissue of the parents
when pecking, and the content of Aloxe3 is reduced during the feeding
period, which is consistent with Aloxe3 leads to a large amount of skin
permeability and water loss leading to death after birth (Fournier et
al., 2019).
PTPRJ a receptor-like protein tyrosine phosphatase (PTP) is expressed in
several cell types. It is initially identified as a negative regulator
of cell proliferation, and consistent with its increased expression in
confluent epithelial and endothelial cells (Spring et al., 2012).
Meanwhile, it has been suggested to contribute to cell-cell contact
inhibition (Fournier, Dussault, Fusco, Rivard, & Royal, 2016). Many of
its identified substrates are growth factor receptors, including VEGFR2,
as well as ERK1/2 and cell adhesion proteins (Spring et al., 2012).
PTPRJ dephosphorylates several
growth factors and their receptors, negatively regulating cell
proliferation and migration (Chabot, Spring, Gratton, Elchebly, &
Royal, 2009). PTPRJ acts as an important regulator of Notch signaling
and endothelial sprouting during retinal vascular development.
Recent evidence suggests that the
protein tyrosine phosphatase PTPRJ ACTS as a promoter of angiogenesis
both in vivo and in vitro. There are abundant and dense capillaries on
the skin surface of discus fish. The larvae absorb nutrients by biting
the skin of the parent fish. We speculate that the nutrients absorbed by
the larvae may be provided by the substances in capillaries in the skin
of the parents of discus fish (Ghavami et al., 2009; Hashemi,
Moazeni‐Roodi, & Ghavami, 2019; Nagata, 2010; Spring, Lapointe, Caron,
Langlois, & Royal, 2014). PTPRJ is the gene that we’re going to focus
on next.
Caspase 3 encoded by CASP3 gene located on the long arm of the
chromosome. CASP3, an effector caspase, is activated by extrinsic and
intrinsic apoptosis pathway and plays a key role in the execution phase
of apoptosis (Nagata, 2010). Caspases, cysteine aspartate specific
proteases, are involved in signaling and execution in apoptosis
pathways. They can be generally divided into initiator and effector
caspases based on their functions (Schorpp-Kistner, Wang, Angel, &
Wagner, 1999). Apoptosis is a genetically programmed cell death process
used to eliminate cells that are injured, infected, or have reached the
end of their lifespan (Bender et al., 2008).The larva of discus fish
pecked the parent’s skin during lactation, which may damage the tissue
cells of the skin and accelerate the rate of cell apoptosis. Therefore,
it was speculated that Caspase 3 might be involved in the lactation
process of discus fish.
JUNB is defined as a member of transcription factor family activator
protein and a critical regulator and expression of inflammatory
modulators in fibroblasts and T lymphocytes (Popov et al., 2011). The
AP-1 transcription factor JUNB,
which is encoded by the immediate early gene JUNB, mediates multiple
biological pro-cesses including placentation, suppression of myeloid
cell proliferation, and maintenance of bone and skin homeostasis. During
lactation, the skin surface of discus fish is unstable, which may be
related to the presence of JUNB and its decreased content. JUNB is a
basic leucine zipper (bZIP) protein and forms an AP-1complex by
dimerizing with other bZIP proteins, such as Fos or BATF family members
(Puthiyaveetil, Kota, Chakkarayan, Chakkarayan, & Thodiyil, 2016;
Sadri, Farhadi, & Nourmohamadi, 2019). It has been shown that JUNB is
induced by ALK-NPM, participating the mTOR pathway (Yoo et al., 2018).
In addition, JUNB acts as a regulator of vascular endothelial growth
factor (VEGF) protein production and affects vessel proliferation and
tissue angiogenesis. and acts as a VEGF production protein regulator and
influences vessel proliferation and tissue angiogenesis (Popov et al.,
2011). VEGF is a multifunctional cytokine that expresses in different
situations and has a role in increasing vascular permeability and
angiogenesis, thereby stimulating the proliferation and migration of
endothelial cells (Bornstein, Agah, & Kyriakides, 2005). It’s been
reported that the lack of JUNB in different types of cells leads to very
weak VEGF expression and postponed cell growth (Bornstein et al., 2005).
During lactation, the expression of JUNB in larva of discus fish is
decreased (Fig. 6.A), which may be caused by skin damage of discus fish.
The ITGA11 gene has been localized in bands q22.3 q23 on chromosome 15,
and the gene encodes a mature protein with a large 1120-residue
extracellular domain that contains an I-domain of 207 residues and is
linked by a transmembrane domain to a short cytoplasmic domain of 24
amino acids (K. Lehnert et al., 1999). ITGA11 up-regulation has been
shown to be dependent of TGFb-mediated signaling. The ITGA11 gene
methylation status in the amnion may be valuable in explaining the
mechanism of preterm birth (Klaus Lehnert et al., 1999). Generally,
integrins and thrombospondins are known to play an important role for
the regulation of cellular processes, such as cell adhesion, migration,
and differentiation (Ji et al.,
2011). ITGA11 gene expression is
also increased through mid-gestation and decreased through the late
stages of pregnancy (Yoo et al., 2018). This is consistent with
increased expression of the ITGA11 gene during the breeding of discus
(Fig 6 A). We speculate that the lactation process of angelfish is
equivalent to the process of mammalian pregnancy, and the change of
ITGA11 gene in P_S further confirms our speculation.
PRKCD, also known as protein kinase Cδ, was a protein kinase C isozyme
that does not require calcium for its activity (Gschwendt, 1999).
Activation of PPKCD was known to be associated with inhibition of cell
cycle progression, suggesting that PRKCD had a negative effect on cell
survival. Reported of PRKCD had been reported to be associated with
apoptosis induced by various DNA damaging agents, including UV
radiation, ionizing radiation, cisplatin, etoposide, cytosine
arabinoside, mitomycin C and doxorubicin (Ke et al., 2013). During the
two periods of discus fish, we found that P_B compared with NP_B, the
content of PRKCD tended to decrease. It is possible that during
lactation, the young fish will peck at the parent’s skin, shortening the
parent’s skin cell cycle and resulting in a decrease in
PRKCD content. PRKCD regulates
membrane excitability by modulating different ion channels and pumps
such as the calcium efflux regulator PMCA (plasma membrane calcium
ATPase). In the skin, if the permeability barrier is disrupted by
physical or mechanical damage, there is an increase on trans-epidermal
water loss, followed by a decrease of the extracellular
Ca2+ levels. The
decrease in Ca2+ levels causes the opening of calcium
channels allowing calcium influx to restore basal levels of
Ca2+. Accumulation of Ca2+ in the
cell causes the activation of phospholipase C (PLCγ). The activated
phospholipase cleaves phosphatidylinositol 4,5-biphosphate (PI (4,5) P2)
generating DAG and inositol 1,4,5-triphosphate which in turn activate
PRKCD. Thus, PRKCD is fundamental to maintain Ca2+levels and the homeostatic balance in the skin (Malavez, Gonzalez-Mejia,
& Doseff, 2009). During lactation, the discus fish pecked at the
parents’ skin, causing physical damage to the parents’ skin and losing
the balance of moisture and Ca2 + in the skin. This is
consistent with the changes in PRKCD content caused by damage to the
parents’ skin during lactation of discus fish.
The process of feeding the larvae or pecking the parents of discus fish
is a process of information exchange, and the external environment of
the skin of discus fish also changes during the process of lactation and
the discus brain has a neural regulation of environmental change, we
found that the expression of the H1.5 gene changed during lactation. The
H1.5-boundgenes function mainly in cell-cell communication and/or
response to the environment. Linker histone H1.5 belongs to a family of
proteins that organize eukaryotic DNA into a compact structure. The
post-translational modifications induce conformational changes and allow
the nuclear proteins to interact with chromatin which results in the
regulation of transcription and gene expression during cell cycle (Ahmad
et al., 2007). H1.5 forms blocks of chromatin binding in genic and
intergenic regions in differentiated human cells from all germ layers
but not in embryonic stem cells and it has a dynamic distribution during
human cell differentiation and is required for maintenance of proper
gene expression in differentiated cells (J.-Y. Li, Patterson, Mikkola,
Lowry, & Kurdistani, 2012).
OTX2 gene encodes a member of the bicoid subfamily of
homeodomain-containing transcription factors. The encoded protein acts
as a transcription factor and plays a role in brain, craniofacial, and
sensory organ development. The encoded protein also influences the
proliferation and differentiation of dopaminergic neuronal progenitor
cells during mitosis
(https://www.gcbi.com.cn/gclib/html/dictSearch/T1RY- Mg). The milking
behavior of discus fish is very similar to the feeding behavior of
mammals, because the dopamine in mammals increases during reproduction,
so it is possible that the dopamine in discus fish also increases during
milking. So it’s not surprising that increased OTX2 secretion was
detected in the brain during the nurturing period.
HBE is downregulated when P_B compared with NP_B in discus fish brain
(Fig. 6.B). However, it is the same in the skin as in the brain. HBE is
a variant caused by a single point mutation at codon 26 of the b-globin
gene, which is located on chromosome 11p15.5 (Ha, Martinson, Iwamoto, &
Nishi, 2019). People possessing an HBE variant may also develop
secondary disorders like jaun-dice, hepatosplenomegaly and growth
retardation in their devel-opmental stages, which leads to the diagnosis
of HBE (Fucharoen & Weatherall, 2012). Elizabeth Greene,
Joshua Flees et al found the circulatory- and breast muscle-oxygen
homeostasis is dysregulated [low oxygen and hemoglobin (HB) levels]
in chickens with WB myopathy. Molecular analysis showed that blood HB
subunit Mu (HBM), Zeta (HBZ), HBE and hephaestin (HEPH) expression were
significantly down regulated (Fucharoen & Weatherall, 2012).
Interestingly, this result was consistent with the downregulation of the
relative expression of HBE in the lactation behavior of discus fish,
suggesting that lactation behavior of discus fish is similar to that of
mammals. HBE is the gene that we’re going to focus on next.
This study has some limitations. First, the function of the genes
screened by the transcriptome in the parental discus fish unclear.
Second, the parental care process of discus fish is similar to that of
mammals, but very different. Therefore, it is urgent to continue to
explore the parental care process of discus fish.