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.