Differentiation In Zebrafish Research Articles

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres.

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Sexual disruption in zebrafish (Danio rerio) exposed to mixtures of 17α-ethinylestradiol and 17β-trenbolone.

Environmental estrogens and androgens can be present simultaneously in aquatic environments and thereby interact to disturb multiple physiological systems in organisms. Studies on interaction effects in fish of androgenic and estrogenic chemicals are limited. Therefore, the aim of the present study was to evaluate feminization and masculinization effects in zebrafish (Danio rerio) exposed to combinations of two synthetic steroid hormones detected in environmental waters: the androgen 17β-trenbolone (Tb) and the oestrogen 17α-ethinylestradiol (EE2). Juvenile zebrafish were exposed between days 20 and 60 post-hatch to different binary mixtures of Tb (1, 10, and 50 ng/L) and EE2 (2 and 5 ng/L). The endpoints studied were whole-body homogenate vitellogenin concentration at 40 days post-hatch, and sex ratio including gonad maturation at 60 days post-hatch. The feminizing potency of 5 ng/L of EE2, alone as well as in combination with Tb, was clear in the present study, with exposures resulting in almost all-female populations and females being sexually immature. Masculinization effects with male-biased sex ratios were observed when fish were exposed to 2 ng/L of EE2 in combination with Tb concentrations. Intersex fish were observed after exposure to mixtures of 2 ng/L EE2 with 50 ng/L Tb. Sexual maturity generally increased among males at increasing concentrations of Tb. The results of the present study show that exposure to environmentally relevant mixtures of an oestrogen and androgen affects the process of gonad differentiation in zebrafish and lead to sexual disruption.

Fsh Stimulates Spermatogonial Proliferation and Differentiation in Zebrafish via Igf3.

Growth factors modulate germ line stem cell self-renewal and differentiation behavior. We investigate the effects of Igf3, a fish-specific member of the igf family. Fsh increased in a steroid-independent manner the number and mitotic index of single type A undifferentiated spermatogonia and of clones of type A differentiating spermatogonia in adult zebrafish testis. All 4 igf gene family members in zebrafish are expressed in the testis but in tissue culture only igf3 transcript levels increased in response to recombinant zebrafish Fsh. This occurred in a cAMP/protein kinase A-dependent manner, in line with the results of studies on the igf3 gene promoter. Igf3 protein was detected in Sertoli cells. Recombinant zebrafish Igf3 increased the mitotic index of type A undifferentiated and type A differentiating spermatogonia and up-regulated the expression of genes related to spermatogonial differentiation and entry into meiosis, but Igf3 did not modulate testicular androgen release. An Igf receptor inhibitor blocked these effects of Igf3. Importantly, the Igf receptor inhibitor also blocked Fsh-induced spermatogonial proliferation. We conclude that Fsh stimulated Sertoli cell production of Igf3, which promoted via Igf receptor signaling spermatogonial proliferation and differentiation and their entry into meiosis. Because previous work showed that Fsh also released spermatogonia from an inhibitory signal by down-regulating anti-Müllerian hormone and by stimulating androgen production, we can now present a model, in which Fsh orchestrates the activity of stimulatory (Igf3, androgens) and inhibitory (anti-Müllerian hormone) signals to promote spermatogenesis.

The progestin levonorgestrel affects sex differentiation in zebrafish at environmentally relevant concentrations

Synthetic progestins have become widespread environmental contaminants and may cause adverse effects on fish. In the present study, we investigated the effects of levonorgestrel (LNG) on sex differentiation in zebrafish (Danio rerio). Embryos were exposed to LNG at environmentally relevant concentrations (0, 1, 10, 33, and 100ng/L) and allowed to develop until sexual maturity. Histological examination at 63 days post fertilization (dpf) caused complete sex reversal and 100% males were observed in the 10, 33 and 100ng/L treatments; gross morphological and histological examination of gonads at 142dpf further confirmed 100% males at these exposure concentrations. The results indicate androgenic activity of LNG, and masculinization during zebrafish gonadal differentiation. The mRNA expression levels of genes involved in fish sex differentiation and gonadal development were examined at 28 and 42dpf. Down-regulation of the mRNA expression of aromatase (e.g., cyp19a1a, cyp19a1b), the forkhead transcription factor gene L2 (foxl2) and the Fushi tarazu factor-1d (nr5a1b) were observed. In contrast, transcription of the doublesex and mab-3-related transcription factor 1 (dmrt1) gene was up-regulated. Androgen receptor (ar) mRNA expression was significantly down-regulated at 28 and 42dpf. Co-exposure to flutamide (an androgen antagonist) and LNG, led to a decrease in the sex inversion potency of LNG. Our study has demonstrated that environmentally relevant concentrations of LNG could alter sex differentiation and gonadal development in zebrafish. Our results also suggest a potentially high ecological risk of LNG to fish populations in LNG-contaminated aquatic environments.

Long-term exposure to environmentally relevant concentrations of progesterone and norgestrel affects sex differentiation in zebrafish (Danio rerio)

The aim of this study was to investigate the effects of progestins on the sex differentiation of zebrafish by measuring the sex ratio and transcriptions of genes related to sex differentiation (Amh, Dmrt1, Figa, Sox9a and Sox9b genes) as well as sex hormone levels and transcriptional expression profiles along the hypothalamic–pituitary–gonadal (HPG) and hypothalamic–pituitary–adrenal (HPA) axes in juvenile zebrafish. Exposure of zebrafish to 4, 33, 63ngL−1 progesterone (P4) or 4, 34, 77ngL−1 norgestrel (NGT) started at 20 days post fertilization (dpf) and ended at 60 dpf. The results showed that exposure to P4 caused a significant increase in proportion of females as well as significant down-regulation of Amh gene and up-regulation of Figa at a concentration of 63ngL−1. However, the shift in the sex ratio toward males was observed following exposure to 34 and 77ngL−1 NGT, which came along with the significant induction of Dmrt1 gene and inhibition of Figa gene. The sex hormones in exposed fish were measured with estrone being detected only in the fish exposed to the highest P4 concentration; whereas estradiol and androstenedione were detected only in the fish of the control and lowest NGT concentration. Furthermore, the increase in females was associated with the significant up-regulation of several key genes controlling the synthesis of sex hormones (i.e., Cyp17, Cyp19a1a and Hsd3b) following exposure to 63ngL−1 P4 whereas the significant down-regulation of Cyp11a1, Cyp17, Cyp19a1a and Hsd3b genes was observed in the male-biased populations caused by 34 and 77ngL−1 NGT. The overall results imply that both P4 and NGT could significantly affect sex differentiation in zebrafish, and that changes may be reflected by altered sex hormone levels and transcriptional expression profiles of genes related to synthesis of sex hormones.

Pbx4 is Required for the Temporal Onset of Zebrafish Myocardial Differentiation

Proper control of the temporal onset of cellular differentiation is critical for regulating cell lineage decisions and morphogenesis during development. Pbx homeodomain transcription factors have emerged as important regulators of cellular differentiation. We previously showed, by using antisense morpholino knockdown, that Pbx factors are needed for the timely activation of myocardial differentiation in zebrafish. In order to gain further insight into the roles of Pbx factors in heart development, we show here that zebrafish pbx4 mutant embryos exhibit delayed onset of myocardial differentiation, such as delayed activation of tnnt2a expression in early cardiomyocytes in the anterior lateral plate mesoderm. We also observe delayed myocardial morphogenesis and dysmorphic patterning of the ventricle and atrium, consistent with our previous Pbx knock-down studies. In addition, we find that pbx4 mutant larvae have aberrant outflow tracts and defective expression of the proepicardial marker tbx18. Finally, we present evidence for Pbx expression in cardiomyocyte precursors as well as heterogeneous Pbx expression among the pan-cytokeratin-expressing proepicardial cells near the developing ventricle. In summary, our data show that Pbx4 is required for the proper temporal activation of myocardial differentiation and establish a basis for studying additional roles of Pbx factors in heart development.

Early Depletion of Primordial Germ Cells in Zebrafish Promotes Testis Formation

SummaryAs complete absence of germ cells leads to sterile males in zebrafish, we explored the relationship between primordial germ cell (PGC) number and sexual development. Our results revealed dimorphic proliferation of PGCs in the early zebrafish larvae, marking the beginning of sexual differentiation. We applied morpholino-based gene knockdown and cell transplantation strategies to demonstrate that a threshold number of PGCs is required for the stability of ovarian fate. Using histology and transcriptomic analyses, we determined that zebrafish gonads are in a meiotic ovarian stage at 14 days postfertilization and identified signaling pathways supporting meiotic oocyte differentiation and eventual female fate. The development of PGC-depleted gonads appears to be restrained and delayed, suggesting that PGC number may directly regulate the variability and length of gonadal transformation and testicular differentiation in zebrafish. We propose that gonadal transformation may function as a developmental buffering mechanism to ensure the reproductive outcome.

Whole-genome expression profile in zebrafish embryos after chronic exposure to morphine: identification of new genes associated with neuronal function and mu opioid receptor expression.

BackgroundA great number of studies have investigated changes induced by morphine exposure in gene expression using several experimental models. In this study, we examined gene expression changes during chronic exposure to morphine during maturation and differentiation of zebrafish CNS.ResultsMicroarray analysis showed 254 genes whose expression was identified as different by at least 1.3 fold change following chronic morphine exposure as compared to controls. Of these, several novel genes (grb2, copb2, otpb, magi1b, grik-l, bnip4 and sox19b) have been detected for the first time in an experimental animal model treated with morphine. We have also identified a subset of genes (dao.1, wls, bnip4 and camk1γb) differentially expressed by chronic morphine exposure whose expression is related to mu opioid receptor gene expression. Altered expression of copb2, bnip4, sox19b, otpb, dao.1, grik-l and wls is indicative of modified neuronal development, CNS patterning processes, differentiation and dopaminergic neurotransmission, serotonergic signaling pathway, and glutamatergic neurotransmission. The deregulation of camk1γb signaling genes suggests an activation of axonogenesis and dendritogenesis.ConclusionsOur study identified different functional classes of genes and individual candidates involved in the mechanisms underlying susceptibility to morphine actions related to CNS development. These results open new lines to study the treatment of pain and the molecular mechanisms involved in addiction. We also found a set of zebrafish-specific morphine-induced genes, which may be putative targets in human models for addiction and pain processes.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-874) contains supplementary material, which is available to authorized users.

SCUBE3 (Signal Peptide-CUB-EGF Domain-containing Protein 3) Modulates Fibroblast Growth Factor Signaling during Fast Muscle Development

SCUBE3 (signal peptide CUB-EGF-like domain-containing protein 3) belongs to a newly identified secreted and cell membrane-associated SCUBE family, which is evolutionarily conserved in vertebrates. Scube3 is predominantly expressed in a variety of developing tissues in mice such as somites, neural tubes, and limb buds. However, its function during development remains unclear. In this study, we first showed that knockdown of SCUBE3 in C2C12 myoblasts inhibited FGF receptor 4 expression and FGF signaling, thus resulting in reduced myogenic differentiation. Furthermore, knockdown of zebrafish scube3 by antisense morpholino oligonucleotides specifically suppressed the expression of the myogenic marker myod1 within the lateral fast muscle precursors, whereas its expression in the adaxial slow muscle precursors was largely unaffected. Consistent with these findings, immunofluorescent staining of fast but not slow muscle myosin was markedly decreased in scube3 morphants. Further genetic studies identified fgf8 as a key regulator in scube3-mediated fast muscle differentiation in zebrafish. Biochemical and molecular analysis showed that SCUBE3 acts as a FGF co-receptor to augment FGF8 signaling. Scube3 may be a critical upstream regulator of fast fiber myogenesis by modulating fgf8 signaling during zebrafish embryogenesis.

The Hippo pathway controls a switch between retinal progenitor cell proliferation and photoreceptor cell differentiation in zebrafish.

The precise regulation of numbers and types of neurons through control of cell cycle exit and terminal differentiation is an essential aspect of neurogenesis. The Hippo signaling pathway has recently been identified as playing a crucial role in promoting cell cycle exit and terminal differentiation in multiple types of stem cells, including in retinal progenitor cells. When Hippo signaling is activated, the core Mst1/2 kinases activate the Lats1/2 kinases, which in turn phosphorylate and inhibit the transcriptional cofactor Yap. During mouse retinogenesis, overexpression of Yap prolongs progenitor cell proliferation, whereas inhibition of Yap decreases this proliferation and promotes retinal cell differentiation. However, to date, it remains unknown how the Hippo pathway affects the differentiation of distinct neuronal cell types such as photoreceptor cells. In this study, we investigated whether Hippo signaling regulates retinogenesis during early zebrafish development. Knockdown of zebrafish mst2 induced early embryonic defects, including altered retinal pigmentation and morphogenesis. Similar abnormal retinal phenotypes were observed in zebrafish embryos injected with a constitutively active form of yap [(yap (5SA)]. Loss of Yap’s TEAD-binding domain, two WW domains, or transcription activation domain attenuated the retinal abnormalities induced by yap (5SA), indicating that all of these domains contribute to normal retinal development. Remarkably, yap (5SA)-expressing zebrafish embryos displayed decreased expression of transcription factors such as otx5 and crx, which orchestrate photoreceptor cell differentiation by activating the expression of rhodopsin and other photoreceptor cell genes. Co-immunoprecipitation experiments revealed that Rx1 is a novel interacting partner of Yap that regulates photoreceptor cell differentiation. Our results suggest that Yap suppresses the differentiation of photoreceptor cells from retinal progenitor cells by repressing Rx1-mediated transactivation of photoreceptor cell genes during zebrafish retinogenesis.

SoxF factors and Notch regulate nr2f2 gene expression during venous differentiation in zebrafish

Initial embryonic determination of artery or vein identity is regulated by genetic factors that work in concert to specify the endothelial cell׳s (EC) fate, giving rise to two structurally unique components of the circulatory loop. The Shh/VEGF/Notch pathway is critical for arterial specification, while the orphan receptor nr2f2 (COUP-TFII) has been implicated in venous specification. Studies in mice have shown that nr2f2 is expressed in venous but not arterial ECs, and that it preferentially induces markers of venous cell fate. We have examined the role of nr2f2 during early arterial–venous development in the zebrafish trunk. We show that expression of a subset of markers of venous endothelial identity requires nr2f2, while the expression of nr2f2 itself requires sox7 and sox18 gene function. However, while sox7 and sox18 are expressed in both the cardinal vein and the dorsal aorta during early trunk development, nr2f2 is expressed only in the cardinal vein. We show that Notch signaling activity present in the dorsal aorta suppresses expression of nr2f2, restricting nr2f2-dependent promotion of venous differentiation to the cardinal vein.

Hyaluronic Acid Receptor Stabilin-2 Regulates Erk Phosphorylation and Arterial - Venous Differentiation in Zebrafish

The hyaluronic acid receptor for endocytosis Stabilin-2/HARE mediates systemic clearance of multiple glycosaminoglycans from the vascular and lymphatic circulations. In addition, recent in vitro studies indicate that Stab2 can participate in signal transduction by interacting with hyaluronic acid (HA), which results in Erk phosphorylation. However, it is not known whether Stab2 function or HA-Stab2 signaling play any role in embryonic development. Here we show that Stab2 functions in a signal transduction pathway regulating arterial-venous differentiation during zebrafish embryogenesis. Stab2 morpholino knockdown embryos (morphants) display an absence of intersegmental vessels and defects in the axial vessel formation. In addition, Stab2 morphants show defects in arterial-venous differentiation including the expansion of venous marker expression. Simultaneous knockdown of Stabilin-2 and Has2, an HA synthetase, results in a synergistic effect, arguing that HA and Stab2 interact during vasculature formation. Stab2 morphants display reduced Erk phosphorylation in the arterial progenitors, which is a known transducer of VEGF signaling, previously associated with arterial-venous differentiation. In addition, VEGF signaling acts as a negative feedback loop to repress stab2 expression. These results argue that Stab2 is involved in a novel signaling pathway that plays an important role in regulating Erk phosphorylation and establishing arterial-venous identity.

Gonad Differentiation in Zebrafish Is Regulated by the Canonical Wnt Signaling Pathway1

Zebrafish males undergo a "juvenile ovary-to-testis" gonadal transformation process. Several genes, including nuclear receptor subfamily 5, group A (nr5a) and anti-Müllerian hormone (amh), and pathways such as Tp53-mediated germ-cell apoptosis have been implicated in zebrafish testis formation. However, our knowledge of the regulation of this complex process is incomplete, and much remains to be investigated about the molecular pathways and network of genes that control it. Using a microarray-based analysis of transforming zebrafish male gonads, we demonstrated that their transcriptomes undergo transition from an ovary-like pattern to an ovotestis to a testis-like profile. Microarray results also validated the previous histological and immunohistochemical observation that there is high variation in the duration and extent of commitment to the juvenile ovary phase among individuals. Interestingly, global gene expression profiling of diverging zebrafish juvenile ovaries and transforming ovotestes revealed that some members of the canonical Wnt/beta-catenin signaling pathway were differentially expressed between these two phases. To investigate whether Wnt/beta-catenin signaling plays a role in zebrafish gonad differentiation, we used the Tg (hsp70l:dkk1b-GFP)w32 line to inhibit Wnt/beta-catenin signaling during gonad differentiation. Activation of dkk1b-GFP expression by heat shock resulted in an increased proportion of males and corresponding decrease in gonadal aromatase gene (cyp19a1a) expression. The Wnt target gene, lymphocyte enhancer binding factor 1 (lef1), was also down-regulated in the process. Together, these results provide the first functional evidence that, similarly to mammals, Wnt/beta-catenin signaling is a "pro-female" pathway that regulates gonad differentiation in zebrafish.

Overnutrition induces β-cell differentiation through prolonged activation of β-cells in zebrafish larvae

Insulin from islet β-cells maintains glucose homeostasis by stimulating peripheral tissues to remove glucose from circulation. Persistent elevation of insulin demand increases β-cell number through self-replication or differentiation (neogenesis) as part of a compensatory response. However, it is not well understood how a persistent increase in insulin demand is detected. We have previously demonstrated that a persistent increase in insulin demand by overnutrition induces compensatory β-cell differentiation in zebrafish. Here, we use a series of pharmacological and genetic analyses to show that prolonged stimulation of existing β-cells is necessary and sufficient for this compensatory response. In the absence of feeding, tonic, but not intermittent, pharmacological activation of β-cell secretion was sufficient to induce β-cell differentiation. Conversely, drugs that block β-cell secretion, including an ATP-sensitive potassium (K ATP) channel agonist and an L-type Ca(2+) channel blocker, suppressed overnutrition-induced β-cell differentiation. Genetic experiments specifically targeting β-cells confirm existing β-cells as the overnutrition sensor. First, inducible expression of a constitutively active K ATP channel in β-cells suppressed the overnutrition effect. Second, inducible expression of a dominant-negative K ATP mutant induced β-cell differentiation independent of nutrients. Third, sensitizing β-cell metabolism by transgenic expression of a hyperactive glucokinase potentiated differentiation. Finally, ablation of the existing β-cells abolished the differentiation response. Taken together, these data establish that overnutrition induces β-cell differentiation in larval zebrafish through prolonged activation of β-cells. These findings demonstrate an essential role for existing β-cells in sensing overnutrition and compensating for their own insufficiency by recruiting additional β-cells.

Downregulation of Max dimerization protein 3 is involved in decreased visceral adipose tissue by inhibiting adipocyte differentiation in zebrafish and mice

The diet-induced obesity model of zebrafish (DIO-zebrafish) share a common pathophysiological pathway with mammalian obesity. We aimed to investigate the role of Max dimerization protein 3 (MXD3) in visceral fat accumulation and adipocyte differentiation, by conducting knockdown experiments using zebrafish and mouse preadipocytes. To identify genes related to visceral adiposity, we conducted transcriptome analyses of human and zebrafish obese populations using the Gene Expression Omnibus and DNA microarray. We then intraperitoneally injected morpholino antisense oligonucleotides (MO-mxd3) to knockdown mxd3 gene expression in DIO-zebrafish and measured several parameters, which reflected human obesity and associated metabolic diseases. Finally, lentiviral Mxd3 shRNA knockdown in mouse 3T3-L1 preadipocytes was conducted. Quantitative PCR analyses of several differentiation markers were conducted during these gene knockdown experiments. We found that MXD3 expression was increased in the obese population in humans and zebrafish. Intraperitoneal MO-mxd3 administration to DIO-zebrafish suppressed the increase in body weight, visceral fat accumulation and the size of mature adipocytes. Subsequently, dyslipidemia and liver steatosis were also ameliorated by MO-mxd3. In mouse adipocytes, Mxd3 expression was drastically increased in the early differentiation stage. Mxd3 shRNA inhibited preadipocyte proliferation and adipocyte maturation. Quantitative PCR analyses showed that the early differentiation marker, CCAAT/enhancer-binding protein delta (Cebpd) and late differentiation markers (CCAAT/enhancer-binding protein, alpha and peroxisome proliferator-activated receptor gamma) were downregulated by Mxd3 knockdown in 3T3-L1 cells and DIO-zebrafish. Subsequently, mature adipocyte markers (adiponectin and caveolin 1 for zebrafish, and fatty acid binding protein 4 and stearoyl-coenzyme A desaturase 1 for mouse adipocytes) were also decreased. Mxd3 regulates preadipocyte proliferation and early adipocyte differentiation via Cebpd downregulation in vitro and in vivo. Integrated analysis of human and zebrafish transcriptomes allows identification of a novel therapeutic target against human obesity and further associated metabolic disease.

Knockdown of Lingo1b protein promotes myelination and oligodendrocyte differentiation in zebrafish

Demyelinating diseases include multiple sclerosis, which is a neurodegenerative disease characterized by immune attacks on the central nervous system (CNS), resulting in myelin sheath damage and axonal loss. Leucine-rich repeat and immunoglobulin domain-containing neurite outgrowth inhibitory protein (Nogo) receptor-interacting protein-1 (LINGO-1) have been identified as a negative regulator of oligodendrocytes differentiation. Targeted LINGO-1 inhibition promotes neuron survival, axon regeneration, oligodendrocyte differentiation, and remyelination in diverse animal models. Although studies in rodent models have extended our understanding of LINGO-1, its roles in neural development and myelination in zebrafish (Danio rerio) are not yet clear. In this study, we cloned the zebrafish homolog of the human LINGO-1 and found that lingo1b regulated myelination and oligodendrocyte differentiation. The expression of lingo1b started 1 (mRNA) and 2 (protein) days post-fertilization (dpf) in the CNS. Morpholino oligonucleotide knockdown of lingo1b resulted in developmental abnormalities, including less dark pigment, small eyes, and a curly spinal cord. The lack of lingo1b enhanced myelination and oligodendrocyte differentiation during embryogenesis. Furthermore, immunohistochemistry and movement analysis showed that lingo1b was involved in the axon development of primary motor neurons. These results suggested that Lingo1b protein functions as a negative regulator of myelination and oligodendrocyte differentiation during zebrafish development.

Aldh1-Expressing Endocrine Progenitor Cells Regulate Secondary Islet Formation in Larval Zebrafish Pancreas

Aldh1 expression is known to mark candidate progenitor populations in adult and embryonic mouse pancreas, and Aldh1 enzymatic activity has been identified as a potent regulator of pancreatic endocrine differentiation in zebrafish. However, the location and identity of Aldh1-expressing cells in zebrafish pancreas remain unknown. In this study we demonstrate that Aldh1-expressing cells are located immediately adjacent to 2F11-positive pancreatic ductal epithelial cells, and that their abundance dramatically increases during zebrafish secondary islet formation. These cells also express neurod, a marker of endocrine progenitor cells, but do not express markers of more mature endocrine cells such as pax6b or insulin. Using formal cre/lox-based lineage tracing, we further show that Aldh1-expressing pancreatic epithelial cells are the direct progeny of pancreatic notch-responsive progenitor cells, identifying them as a critical intermediate between multi-lineage progenitors and mature endocrine cells. Pharmacologic manipulation of Aldh1 enzymatic activity accelerates cell entry into the Aldh1-expressing endocrine progenitor pool, and also leads to the premature maturation of these cells, as evidenced by accelerated pax6b expression. Together, these findings suggest that Aldh1-expressing cells act as both participants and regulators of endocrine differentiation during zebrafish secondary islet formation.

Retinoic Acid Metabolic Genes, Meiosis, and Gonadal Sex Differentiation in Zebrafish

To help understand the elusive mechanisms of zebrafish sex determination, we studied the genetic machinery regulating production and breakdown of retinoic acid (RA) during the onset of meiosis in gonadogenesis. Results uncovered unexpected mechanistic differences between zebrafish and mammals. Conserved synteny and expression analyses revealed that cyp26a1 in zebrafish and its paralog Cyp26b1 in tetrapods independently became the primary genes encoding enzymes available for gonadal RA-degradation, showing lineage-specific subfunctionalization of vertebrate genome duplication (VGD) paralogs. Experiments showed that zebrafish express aldh1a2, which encodes an RA-synthesizing enzyme, in the gonad rather than in the mesonephros as in mouse. Germ cells in bipotential gonads of all zebrafish analyzed were labeled by the early meiotic marker sycp3, suggesting that in zebrafish, the onset of meiosis is not sexually dimorphic as it is in mouse and is independent of Stra8, which is required in mouse but was lost in teleosts. Analysis of dead-end knockdown zebrafish depleted of germ cells revealed the germ cell-independent onset and maintenance of gonadal aldh1a2 and cyp26a1 expression. After meiosis initiated, somatic cell expression of cyp26a1 became sexually dimorphic: up-regulated in testes but not ovaries. Meiotic germ cells expressing the synaptonemal complex gene sycp3 occupied islands of somatic cells that lacked cyp26a1 expression, as predicted by the hypothesis that Cyp26a1 acts as a meiosis-inhibiting factor. Consistent with this hypothesis, females up-regulated cyp26a1 in oocytes that entered prophase-I meiotic arrest, and down-regulated cyp26a1 in oocytes resuming meiosis. Co-expression of cyp26a1 and the pluripotent germ cell stem cell marker pou5f1(oct4) in meiotically arrested oocytes was consistent with roles in mouse to promote germ cell survival and to prevent apoptosis, mechanisms that are central for tipping the sexual fate of gonads towards the female pathway in zebrafish.

Analysis of protein expression in zebrafish during gonad differentiation by targeted proteomics

The molecular mechanisms governing sex determination and differentiation in the zebrafish (Danio rerio) are not fully understood. To gain more insights into the function of specific genes in these complex processes, the expression of multiple candidates needs to be assessed, preferably on the protein level. Here, we developed a targeted proteomics method based on selected reaction monitoring (SRM) to study the candidate sex-related proteins in zebrafish which were selected based on a global proteomics analysis of adult gonads and representational difference analysis of male and female DNA, as well as on published information on zebrafish and other vertebrates. We employed the developed SRM protocols to acquire time-resolved protein expression profiles during the gonad differentiation period in vas::EGFP transgenic zebrafish. Evidence on protein expression was obtained for the first time for several candidate genes previously studied only on the mRNA level or suggested by bioinformatic predictions. Tuba1b (tubulin alpha 1b), initially included in the study as one of the potential housekeeping proteins, was found to be preferentially expressed in the adult testis with nearly absent expression in the ovary. The revealed changes in protein expression patterns associated with gonad differentiation suggest that several of the examined proteins, especially Ilf2 and Ilf3 (interleukin enhancer-binding factors 2 and 3), Raldh3 (retinaldehyde dehydrogenase type 3), Zgc:195027 (low density lipoprotein-related receptor protein 3) and Sept5a (septin 5a), may play a specific role in the sexual differentiation in zebrafish.

The roles of LPA2 on erythrocyte and thrombocyte differentiation in zebrafish

Lysophosphatidic acid (LPA) is a simple lipid growth factor regulates cell proliferation, migration, and survival. It binds to a family of trans‐membrane G‐protein‐coupled receptors. In our previous study, LPA receptors were expressed in hematopoietic stem cells (HSCs) and activation of LPA3 leads to erythropoiesis. However, the roles of LPA2 on erythrocyte differentiation remain unclear. During hematopoiesis processes, erythrocytes and thrombocytes have a common precursor. Therefore, we also attempted to determine whether LPA2 regulates thrombocyte differentiation in zebrafish. Anti‐sence morpholino oligonucleotide of LPA2 was injected into zebrafish embryos at different stages. We used o‐dianisidine staining to determine the levels of hemoglobin expression and Real‐Time PCR for mRNA expression of Gata1, an erythropoietic marker. The expression levels of CD41, a thrombocyte marker, were determined by Real‐Time PCR and in situ hybridization. Our results demonstrated that knocked down of LPA2 enhanced the expression of CD41 while no effect was observed for hemoglobin and Gata1 expression. Therefore, our results suggested that activation of LPA2 may decrease thrombocyte differentiation.