The LHb expression is up-regulated during puberty in female zebrafish. However, the molecular mechanism underlying how LHb expression is regulated during puberty remains largely unknown. In this study, we found that the mRNA expression levels of lhb, fshb and cyp19a1b were up-regulated along with the puberty onset in zebrafish. Among the three nuclear estrogen receptors (nERs), the esr2b is the only type whose expression is significantly up-regulated during puberty onset in the pituitary. However, in situ hybridization results revealed that lhb mRNA was colocalized with esr1 and esr2a but not esr2b. Exposure to estradiol (E2) significantly stimulates LHb expression in both wild-type and kiss1-/-;kiss2-/-;gnrh3-/- triple knockout pubertal zebrafish. Moreover, exposure of cultured pituitary cells to E2 increased the LHb expression, indicating that the estrogenic effect on LHb expression could be acted at the pituitary level. Finally, we cloned and analyzed the promoter of lhb by luciferase assay. Our results indicated that the E2 responsive regions of lhb promoter for ERα and ERβ2 are identical, suggesting that ERα and ERβ2 could bind to the same half ERE region of the promoter of lhb, exhibiting a classical ERE-dependent pathway. In summary, we demonstrate that E2 could directly act on the pituitary level to stimulate LHb transcription during puberty in zebrafish.
The habenula, located on the dorsal thalamic surface, is an emotional and reward processing center. As in the mammalian brain, the zebrafish habenula is divided into dorsal (dHb) and ventral (vHb) subdivisions that project to the interpeduncular nucleus and median raphe (MR) respectively. Previously, we have shown that kisspeptin 1 (Kiss1) expressing in the vHb, regulates the serotonin (5-HT) system in the MR. However, the connectivity between the Kiss1 neurons and the 5-HT system remains unknown. To resolve this issue, we generated a specific antibody against zebrafish Kiss1 receptor (Kiss-R1); using this primary antibody we found intense immunohistochemical labeling in the ventro-anterior corner of the MR (vaMR) but not in 5-HT neurons, suggesting the potential involvement of interneurons in 5-HT modulation by Kiss1. Double-fluorescence labeling showed that the majority of habenular Kiss1 neurons are glutamatergic. In the MR region, Kiss1 fibers were mainly seen in close association with glutamatergic neurons and only scarcely within GABAergic and 5-HT neurons. Our findings indicate that the habenular Kiss1 neurons potentially modulate the 5-HT system primarily through glutamatergic neurotransmission via as yet uncharacterized interneurons. The neuropeptide kisspeptin (Kiss1) play a key role in vertebrate reproduction. We have previously shown modulatory role of habenular Kiss1 in the raphe serotonin (5-HT) systems. This study proposed that the habenular Kiss1 neurons modulate the 5-HT system primarily through glutamatergic neurotransmission, which provides an important insight for understanding of the modulation of 5-HT system by the habenula-raphe pathway.
The present study reported the first pathogenic Aeromonas salmonicida (SRW-OG1) isolated from the warm water fish orange-spotted grouper (Epinephelus coioides), and investigated the function of Aryl hydrocarbon receptor (AhR), a ligand-dependent transcriptional factor which has been recently found to be closely associated with immune response in mammals and E. coioides. Our results showed that AhR was activated by an unknown ligand in the spleen, intestine and macrophages. Meanwhile, ahr1a and ahr1b were significantly increased in the spleen, intestine and macrophages, whereas ahr2 was only increased in the intestine, which indicated that the contribution of AhR2 to the immune response may be less than that of AhR1a and AhR1b. Some key genes involved in the macrophage inflammatory response, bacterial recognition, and intestinal immunity were significantly up-regulated in the SRW-OG1 infected E. coioides. Nevertheless, declining macrophage ROS production and down-regulation of related genes were also observed, suggesting that SRW-OG1 utilized its virulence mechanisms to prevent macrophage ROS production. Furthermore, AhR inhibitor 3', 4'-DMF and the silence of ahr1a or ahr1b significantly rescued the increased IL-1β and IL-8 induced by SRW-OG1 infection, which proved that the induction of IL-1β and IL-8 in E. coioides macrophages was mediated by AhR. However, BPI/LBP, ROS production and related genes were not affected by AhR. The survival rate and immune escape rate of SRW-OG1 in the ahr1a/ahr1b knocked-down and 3', 4'-DMF treated macrophages were significantly increased compared with those in wild type macrophages. Taken together, it was preliminarily confirmed that ahr1a and ahr1b played an important role in the immune response against A. salmonicida SRW-OG1.
The kiss1/gpr54 signaling system is considered to be a critical regulator of reproduction in most vertebrates. However, this presumption has not been tested vigorously in nonmammalian vertebrates. Distinct from mammals, multiple kiss1/gpr54 paralogous genes (kiss/kissr) have been identified in nonmammalian vertebrates, raising the possibility of functional redundancy among these genes. In this study, we have systematically generated the zebrafish kiss1(-/-), kiss2(-/-), and kiss1(-/-);kiss2(-/-) mutant lines as well as the kissr1(-/-), kissr2(-/-), and kissr1(-/-);kissr2(-/-) mutant lines using transcription activator-like effector nucleases. We have demonstrated that spermatogenesis and folliculogenesis as well as reproductive capability are not impaired in all of these 6 mutant lines. Collectively, our results indicate that kiss/kissr signaling is not absolutely required for zebrafish reproduction, suggesting that the kiss/kissr systems play nonessential roles for reproduction in certain nonmammalian vertebrates. These findings also demonstrated that fish and mammals have evolved different strategies for neuroendocrine control of reproduction.
In addition to vision, light information is used to regulate a range of animal physiology. Such nonimage-forming functions of light are mediated by nonvisual photoreceptors expressed in distinct neurons in the retina and the brain in most vertebrates. A nonvisual photoreceptor vertebrate ancient long opsin (VAL-opsin) possesses two functional isoforms in the zebrafish, encoded by valopa and valopb, which has received little attention. To delineate the neurochemical identities of valop cells and to test for colocalization of the valop isoforms, we used in situ hybridization to characterize the expression of the valop genes along with that of neurotransmitters and a neuropeptide known to be present at the sites of valop expression. Double labeling showed that the thalamic valop population coexpresses valopa and valopb. All the thalamic valop cells overlapped with a GABAergic cell mass that continues from the anterior nucleus to the intercalated thalamic nucleus. A novel valopa cell population found in the superior raphe was serotonergic in nature. A valopb cell population in the Edinger-Westphal nucleus was identified as containing thyrotropin-releasing hormone. Valopb cells localized in the hindbrain intermediate reticular formation were noncholinergic in nature (nonmotorneurons). Thus, the presence of valop cell populations in different brain regions with coexpression of neurotransmitters and neuropeptides and the colocalization of valop isoforms in the thalamic cell population indicate regulatory and functional complexity of VAL-opsin in the brain of the zebrafish.
The Kiss1/KISS1 gene has recently been implicated as a potent hypothalamic regulator of reproductive functions, in particular, the onset of puberty in mammals. In zebrafish (Danio rerio), there are two kiss1 homologues (kiss1 and kiss2) expressed in the brain: Kiss2-expressing neurons in the hypothalamic nuclei are considered potent regulators of reproduction, whereas the role of Kiss1-expressing neurons in the habenula remains unknown. We first analyzed the expression of kiss1 mRNA in a transgenic zebrafish, in which the habenula-interpeduncular nucleus (IPN) pathway is labelled with green fluorescent protein, and our application of a biocytin neural tracer into the habenula showed the presence of neuronal projections of Kiss1 neurons to the ventral IPN. Therefore, we speculated that kiss1 neurons might regulate the serotonergic system in the raphe. However, laser microdissection followed by real-time PCR revealed the expression of Kiss1 receptor (kissr1) mRNA in the habenula and the ventral IPN but not in the dorsal IPN or the serotonergic neurons in the raphe nuclei. Dual-fluorescent in situ hybridization revealed the coexpression of kiss1 and kissr1 mRNA in the habenula. Administration of Kiss1 significantly decreased the level of kiss1 mRNA (0.3- to 0.5-fold, P < 0.001), but the level of c-fos mRNA was increased (≈ 3-fold, P < 0.05) in the ventral habenula, suggesting that there is autocrine regulation of the kiss1 gene. Kiss1 administration significantly increased the c-fos mRNA levels in the raphe nuclei (2.5-fold, P < 0.001) and genes involved in the regulation of serotonin levels (pet1 and slc6a4a; 3.3- and 2.2-fold, P < 0.01). These findings suggest that the autocrine-regulated habenular Kiss1 neurons indirectly regulate the serotonergic system in the raphe nuclei through the IPN in the zebrafish.
Newly discovered kisspeptin (metastin), encoded by the Kiss1/KISS1 gene, is considered as a major gatekeeper of puberty through the regulation of GnRH. In the present study, we cloned a novel kisspeptin gene (kiss2) in the zebrafish Danio rerio and the medaka Oryzias latipes, which encodes a sequence of 125 and 115 amino acids, respectively, and its core sequence (FNLNPFGLRF, F-F form) is different from the previously characterized kiss1 (YNLNSFGLRY, Y-Y form). Our in silico data mining shows kiss1 and kiss2 are highly conserved across nonmammalian vertebrate species, and we have identified two putative kisspeptins in the platypus and three forms in Xenopus. In the brain of zebrafish and medaka, in situ hybridization and laser capture microdissection coupled with real-time PCR showed kiss1 mRNA expression in the ventromedial habenula and the periventricular hypothalamic nucleus. The kiss2 mRNA expression was observed in the posterior tuberal nucleus and the periventricular hypothalamic nucleus. Quantitative real-time PCR analysis during zebrafish development showed a significant increase in zebrafish kiss1, kiss2 (P < 0.002), gnrh2, and gnrh3 (P < 0.001) mRNA levels at the start of the pubertal phase and remained high in adulthood. In sexually mature female zebrafish, Kiss2 but not Kiss1 administration significantly increased FSH-beta (2.7-fold, P < 0.05) and LH-beta (8-fold, P < 0.01) mRNA levels in the pituitary. These results suggest that the habenular Kiss1 and the hypothalamic Kiss2 are potential regulators of reproduction including puberty and that Kiss2 is the predominant regulator of gonadotropin synthesis in fish.
Epilepsy is a devastating neurological condition exhibited by repeated spontaneous and unpredictable seizures afflicting around 70 million people globally. The basic pathophysiology of epileptic seizures is still elusive, reflecting an extensive need for further research. Developing a novel animal model is crucial in understanding disease mechanisms as well as in assessing the therapeutic target. Most of the pre-clinical epilepsy research has been focused on rodents. Nevertheless, zebrafish disease models are relevant to human disease pathophysiology hence are gaining increased attention nowadays. The current study for the very first time developed a pilocarpine-induced chronic seizure-like condition in adult zebrafish and investigated the modulation in several neuroinflammatory genes and neurotransmitters after pilocarpine exposures. Seizure score analysis suggests that compared to a single dose, repeated dose pilocarpine produces chronic seizure-like effects maintaining an average seizure score of above 2 each day for a minimum of 10 days. Compared to the single dose pilocarpine treated group, there was increased mRNA expression of HMGB1, TLR4, TNF-α, IL-1, BDNF, CREB-1, and NPY; whereas decreased expression of NF-κB was upon the repeated dose of pilocarpine administration. In addition, the epileptic group demonstrates modulation in neurotransmitters levels such as GABA, Glutamate, and Acetylcholine. Moreover, proteomic profiling of the zebrafish brain from the normal and epileptic groups from LCMS/MS quantification detected 77 and 13 proteins in the normal and epileptic group respectively. Summing up, the current investigation depicted that chemically induced seizures in zebrafish demonstrated behavioral and molecular alterations similar to classical rodent seizure models suggesting the usability of adult zebrafish as a robust model to investigate epileptic seizures.
The habenula is an evolutionarily conserved brain structure, which has recently been implicated in fear memory. In the zebrafish, kisspeptin (Kiss1) is predominantly expressed in the habenula, which has been implicated as a modulator of fear response. Hence, in the present study, we questioned whether Kiss1 has a role in fear memory and morphine-induced fear memory impairment using an odorant cue (alarm substances, AS)-induced fear avoidance paradigm in adult zebrafish, whereby the fear-conditioned memory can be assessed by a change of basal place preference (= avoidance) of fish due to AS-induced fear experience. Subsequently, to examine the possible role of Kiss1 neurons-serotonergic pathway, kiss1 mRNA and serotonin levels were measured. AS exposure triggered fear episodes and fear-conditioned place avoidance. Morphine treatment followed by AS exposure, significantly impaired fear memory with increased time-spent in AS-paired compartment. However, fish administered with Kiss1 (10-21 mol/fish) after morphine treatment had significantly lower kiss1 mRNA levels but retained fear memory. In addition, the total brain serotonin levels were significantly increased in AS- and Kiss1-treated groups as compared to control and morphine treated group. These results suggest that habenular Kiss1 might be involved in consolidation or retrieval of fear memory through the serotonin system.
Large doses of ionizing radiation can damage human tissues. Therefore, there is a need to investigate the radiation effects as well as identify effective and non-toxic radioprotectors. This study evaluated the radioprotective effects of Kelulut honey (KH) from stingless bee (Trigona sp.) on zebrafish (Danio rerio) embryos. Viable zebrafish embryos at 24 hpf were dechorionated and divided into four groups, namely untreated and non-irradiated, untreated and irradiated, KH pre-treatment and amifostine pre-treatment. The embryos were first treated with KH (8 mg/mL) or amifostine (4 mM) before irradiation at doses of 11 Gy to 20 Gy using gamma ray source, caesium-137 (137Cs). Lethality and abnormality analysis were performed on all of the embryos in the study. Immunohistochemistry assay was also performed using selected proteins, namely γ-H2AX and caspase-3, to investigate DNA damages and incidences of apoptosis. KH was found to reduce coagulation effects at up to 20 Gy in the lethality analysis. The embryos developed combinations of abnormality, namely microphthalmia (M), body curvature and microphthalmia (BM), body curvature with microphthalmia and microcephaly (BMC), microphthalmia and pericardial oedema (MO), pericardial oedema (O), microphthalmia with microcephaly and pericardial oedema (MCO) and all of the abnormalities (AA). There were more abnormalities developed from 24 to 72 h (h) post-irradiation in all groups. At 96 h post-irradiation, KH was identified to reduce body curvature effect in the irradiated embryos (up to 16 Gy). γ-H2AX and caspase-3 intensities in the embryos pre-treated with KH were also found to be lower than the untreated group at gamma irradiation doses of 11 Gy to 20 Gy and 11 Gy to 19 Gy, respectively. KH was proven to increase the survival rate of zebrafish embryos and exhibited protection against organ-specific abnormality. KH was also found to possess cellular protective mechanism by reducing DNA damage and apoptosis proteins expression.
Substance P (SP) and neurokinin A (NKA), encoded by TAC1/Tac1 gene are members of the tachykinin family, which exert their neuromodulatory roles in vertebrate reproduction. In mammals, SP and NKA have been shown to regulate gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion via kisspeptin neurons. On the other hand, the role of SP/NKA in the regulation of reproduction in non-mammalian vertebrates is not well known. In the present study, we first localized expression of tac1 mRNA in the brain of male and female zebrafish, Danio rerio. Next, using an antibody against zebrafish tachykinin1 (Tac1), we examined the neural association of SP/NKA neural processes with GnRH3 neurons, and with kisspeptin (kiss2) neurons, in the brains of male and female zebrafish. In situ hybridization showed an apparent male-dominant tac1 expression in the ventral telencephalic area, the anterior and posterior parts of the parvocellular preoptic nucleus, and the suprachiasmatic nucleus. On the other hand, there was female-dominant tac1 expression in the ventral periventricular hypothalamus. Confocal images of double-labeled zebrafish Tac1 and GnRH3 showed associations between Tac1-immunoreactive processes and GnRH3 neurons in the ventral telencephalic area. In contrast, there was no apparent proximity of Tac1 processes to kiss2 mRNA-expressing neurons in the hypothalamus. Lastly, to elucidate possible direct action of SP/NKA on GnRH3 or Kiss2 neurons, expression of SP/NKA receptor, tacr1a mRNA was examined in regions containing GnRH3 or Kiss2 neurons by in situ hybridization. Expression of tacr1a mRNA was seen in several brain regions including the olfactory bulb, preoptic area and hypothalamus, where GnRH3 and Kiss2 cells are present. These results suggest that unlike in mammals, Tac1 may be involved in male reproductive functions via direct action on GnRH3 neurons but independent of kisspeptin in the zebrafish.
This study assessed the cholesterol lowering effect of Pediococcus acidilactici LAB4 and Lactobacillus plantarum LAB12 using adult zebrafish. Animals were fed with a high cholesterol diet (HCD) with/without LAB for seven weeks. Serum and liver cholesterol was quantified using colorimetric and dye staining methods. Expressions of npc1l1 and abca1 in the liver and intestine and appa in the brain were quantified using RT-PCR. Serum and liver cholesterol was significantly lowered in LAB4- and LAB12-fed zebrafish (≤64% and ≤71%, respectively), with reduced liver cholesterol deposition. The cholesterol lowering effect was accompanied by down-regulation of npc1l1 in intestines (≤28.7%), up-regulation of abca1 in the liver (≥30.5%) and down-regulation of appa in the brain (≤24.5%). A moderately strong positive Pearson correlation (r = 0.617, p < 0.01) was found between appa and serum cholesterol. LAB-fed zebrafish exhibited improved spatial learning and memory. LAB4 and LAB12 can be potentially used in preventing hypercholesterolaemia and Alzheimer's diseases.
Zebrafish represents a powerful in vivo model for phenotype-based drug discovery to identify clinically relevant small molecules. By utilizing this model, we evaluated natural product derived compounds that could potentially modulate Notch signaling that is important in both zebrafish embryogenesis and pathogenic in human cancers. A total of 234 compounds were screened using zebrafish embryos and 3 were identified to be conferring phenotypic alterations similar to embryos treated with known Notch inhibitors. Subsequent secondary screens using HEK293T cells overexpressing truncated Notch1 (HEK293TΔE) identified 2 compounds, EDD3 and 3H4MB, to be potential Notch antagonists. Both compounds reduced protein expression of NOTCH1, Notch intracellular domain (NICD) and hairy and enhancer of split-1 (HES1) in HEK293TΔE and downregulated Notch target genes. Importantly, EDD3 treatment of human oral cancer cell lines demonstrated reduction of Notch target proteins and genes. EDD3 also inhibited proliferation and induced G0/G1 cell cycle arrest of ORL-150 cells through inducing p27KIP1. Our data demonstrates the utility of the zebrafish phenotypic screen and identifying EDD3 as a promising Notch antagonist for further development as a novel therapeutic agent.
Ambient light and temperature affect reproductive function by regulating kisspeptin and gonadotrophin-releasing hormone (GnRH) in vertebrates. Melatonin and melatonin receptors, as well as the two-pore domain K+ channel-related K+ (TREK) channels, are affected by light and/or temperature; therefore, these molecules could modulate kisspeptin and GnRH against ambient light and temperature. In this study, we investigated the effect of light and temperature, which affect melatonin levels in gene expression levels of TREK channels, kisspeptin, and GnRH. We first investigated the effects of different light and temperature conditions on brain melatonin concentrations by ELISA. Fish were exposed to either constant darkness, constant light, high temperature (35°C), or low temperature (20°C) for 72 h. Brain melatonin levels were significantly high under constant darkness and high temperature. We further investigated the effects of high brain melatonin levels by constant darkness and high temperature on gene expression levels of melatonin receptors (mt1, mt2, and mel1c), TREK channels (trek1b, trek2a, and trek2b), gnrh3, and kiss2 in the adult zebrafish brain by real-time polymerase chain reaction. Fish were exposed to constant darkness or elevated temperatures (35°C) for 72 h. trek2a, kiss2, and gnrh3 levels were increased under constant darkness. High temperature decreased gene expression levels of mt1, mt2, mel1c, and gnrh3 in the preoptic area, whereas other genes remained unchanged. Melatonin receptors, TREK channels, gnrh3, and kiss2 responded differently under high melatonin conditions. The melatonin receptors and the TREK channels could play roles in the regulation of reproduction by environmental cues, especially ambient light and temperature.
CTP Synthase (CTPS) is a metabolic enzyme that is recognized as a catalyst for nucleotide, phospholipid and sialoglycoprotein production. Though the structural characteristics and regulatory mechanisms of CTPS are well-understood, little is known regarding the extent of its involvement during the early developmental stages of vertebrates. Zebrafish carries two CTPS genes, annotated as ctps1a and ctps1b. Phylogenetic analyses show that both genes had diverged from homologues in the ancestral Actinopterygii, Oreochromis niloticus. Conservation of common CTPS-catalytic regions further establishes that both proteins are likely to be functionally similar to hsaCTPS. Here, we show that ctps1a is more critical throughout the initial period of embryonic development than ctps1b. The effects of concurrent partial knockdown are dependent on ctps1a vs ctps1b dosage ratios. When these are equally attenuated, abnormal phenotypes acquired prior to the pharyngula period disappear in hatchlings (48hpf); however, if either gene is more attenuated than the other, these only become more pronounced in advanced stages. Generally, disruption to normal ctps1a or ctps1b expression levels by morpholinos culminates in the distortion of the early spinal column as well as multiple-tissue oedema. Other effects include slower growth rates, increased mortality rates and impaired structural formation of the young fish's extremities. Embryos grown in DON, a glutamine-analogue drug and CTPS antagonist, also exhibit similar characteristics, thus strengthening the validity of the morpholino-induced phenotypes observed. Together, our results demonstrate the importance of CTPS for the development of zebrafish embryos, as well as a disparity in activity and overall importance amongst isozymes.
kcnk10a has been predicted in zebrafish to be a member of the two-pore domain potassium ion (K+) channel-related K+ (TREK) channel family known as a thermoreceptor. Since reproduction is affected by temperature, Kcnk10a could be involved in the regulation of reproduction. However, expression of kcnk10a in the zebrafish brain and association with reproduction has not been identified. In this study, the full length sequence and localization of kcnk10a in the brain was investigated and gene expressions of the TREK channel family were examined to investigate association with reproduction. We initially identified the full length cDNA sequence of kcnk10a using Rapid Amplification of cDNA Ends and localization in the zebrafish brain using in situ hybridization. Furthermore, we examined the gene expression differences of kcnk2b, kcnk10a and kcnk10b mRNA between genders as well as developmental stages by real-time PCR. The deduced amino acid sequence of the identified kcnk10a mRNA contains highly conserved two pore domains and four transmembrane regions and was higher similarity to zebrafish Kcnk10b than zebrafish Kcnk2a and 2b. kcnk10a mRNA was widely distributed in the brain such as the preoptic area, hypothalamus and the midbrain. kcnk10a mRNA expression exhibited significant difference between mature male and female, and increase during puberty. Kcnk10a could be involved in the regulation of reproductive function.
Gonadotrophin-releasing hormone (GnRH) expression is associated with the two-pore domain potassium ion (K+) channel-related K+ (TREK) channel trek2a expression and melatonin levels. We aimed to investigate correlation of trek2a expression with gnrh3 expression, and regulatory mechanisms of trek2a expression by the melatonin receptor Mt1 and α2-adrenoceptor which are regulated by melatonin. trek2a specific siRNA, Mt1 antagonist luzindole and α2-adrenoceptor antagonist prazosin were administered into the adult zebrafish brain and gene expressions were examined by real-time PCR. trek2a specific siRNA administration significantly reduced expression levels of trek2a, gnrh3 and mt1. Luzindole administration suppressed trek2a and gnrh3 expressions. Prazosin administration reduced trek2a and gnrh3 expressions. It is suggested that Trek2a regulates gnrh3 expression under the control of Mt1 and α2-adrenoceptor.
Despite the known importance of long-chained polyunsaturated fatty acids (LC-PUFA) during development, very little is known about their utilization and biosynthesis during embryogenesis. Combining the advantages of the existence of a complete range of enzymes required for LC-PUFA biosynthesis and the well established developmental biology tools in zebrafish, we examined the expression patterns of three LC-PUFA biosynthesis genes, Elovl2-like elongase (elovl2), Elovl5-like elongase (elovl5) and fatty acyl desaturase (fad) in different zebrafish developmental stages. The presence of all three genes in the brain as early as 24 hours post fertilization (hpf) implies LC-PUFA synthesis activity in the embryonic brain. This expression eventually subsides from 72 hpf onwards, coinciding with the initiation of elovl2 and fad expression in the liver and intestine, 2 organs known to be involved in adult fish LC-PUFA biosynthesis. Collectively, these patterns strongly suggest the necessity for localized production of LC-PUFA in the brain during in early stage embryos prior to the maturation of the liver and intestine. Interestingly, we also showed a specific expression of elovl5 in the proximal convoluted tubule (PCT) of the zebrafish pronephros, suggesting a possible new role for LC-PUFA in kidney development and function.
pbx1, a TALE (three-amino acid loop extension) homeodomain transcription factor, is involved in a diverse range of developmental processes. We examined the expression of pbx1 during zebrafish development by in situ hybridization. pbx1 transcripts could be detected in the central nervous system and pharyngeal arches from 24 hpf onwards. In the swim bladder anlage, pbx1 was detected as early as 28 hpf, making it the earliest known marker for this organ. Morpholino-mediated gene knockdown of pbx1 revealed that the swim bladder failed to inflate, with eventual lethality occurring by 8 dpf. The knockdown of pbx1 did not perturb the expression of prdc and foxA3, with both early swim bladder markers appearing normally at 36 and 48 hpf, respectively. However, the expression of anxa5 was completely abolished by pbx1 knockdown at 60 hpf suggesting that pbx1 may be required during the late stage of swim bladder development.
In Parkinson's disease (PD), several genes have been identified as the PD-related genes, however, the regulatory mechanisms of these gene expressions have not been fully identified. In this study, we investigated the effect of inflammation, one of the major risk factors in PD on expressions of the PD-related genes. Lipopolysaccharide (LPS) was intraperitoneally administered to mature male zebrafish and gene expressions in the brains were examined by real-time PCR. In the inflammation-related genes, expressions of tnfb, il1b and il6 were increased at 2 days post administration in the 10 μg group, and tnfb expression was also increased at 4 days post administration in the 1 μg and 10 μg group. In the PD-related genes, pink1 expression was significantly decreased at 4 days, atp13a2 expression was significantly increased at 7 days, and uchl1 expression was significantly decreased at 7 days. This suggests that pink1, atp13a2 and uchl1 expressions are regulated by inflammation, and this regulatory mechanism might be involved in the progress of PD.