Rubén Marín-Juez

Robotic injection of zebrafish embryos for high-throughput screening in disease models

The increasing use of zebrafish larvae for biomedical research applications is resulting in versatile models for a variety of human diseases. These models exploit the optical transparency of zebrafish larvae and the availability of a large genetic tool box. Here we present detailed protocols for the robotic injection of zebrafish embryos at very high accuracy with a speed of up to 2000 embryos per hour. These protocols are benchmarked for several applications: (1) the injection of DNA for obtaining transgenic animals, (2) the injection of antisense morpholinos that can be used for gene knock-down, (3) the injection of microbes for studying infectious disease, and (4) the injection of human cancer cells as a model for tumor progression. We show examples of how the injected embryos can be screened at high-throughput level using fluorescence analysis. Our methods open up new avenues for the use of zebrafish larvae for large compound screens in the search for new medicines.

Transcriptional and proteomic profiling of flatfish (Solea senegalensis) spermatogenesis

The Senegalese sole (Solea senegalensis) is a marine flatfish of high economic value and a target species for aquaculture. The efforts to reproduce this species in captivity have been hampered by the fact that farmed males (F1) often show lower sperm production and fertilization capacity than wild‐type males (F0). Our knowledge on spermatogenesis is however limited to a few studies. In a previous work, we identified by 2‐D DIGE several potential protein markers in testis for the poor reproductive performance of F1 males. Therefore, the objectives of the present study were, first, to investigate changes in genes and proteins expressed in the testis throughout spermatogenesis in F0 males by using a combination of transcriptomic and proteomic approaches and, second, to further compare the testis proteome between late spermatogenic stages of F0 and F1 fish to identify potential indicators of hampered reproductive performance in F1 fish. We identified approximately 400 genes and 49 proteins that are differentially expressed during the progression of spermatogenesis and that participate in processes such as transcriptional activation, the ubiquitin–proteasome system, sperm maturation and motility or cytoskeletal remodeling. Interestingly, a number of these proteins differed in abundance between F0 and F1 fish, pointing toward alterations in cytoskeleton, sperm motility, the ubiquitin–proteasome system and the redox state during spermiogenesis as possible causes for the decreased fertility of F1 fish.

Molecular identification of genes involved in testicular steroid synthesis and characterization of the response to gonadotropic stimulation in the Senegalese sole (Solea senegalensis) testis

In male teleosts, testicular steroids are essential hormones for the regulation of spermatogenesis and their production is regulated by pituitary gonadotropins. In the Senegalese sole (Solea senegalensis), an economically important flatfish with semi-cystic and asynchronous spermatogenesis, the gonadotropic regulation of spermatogenesis, particularly regarding the production and regulation of testicular steroids, are not well understood. For this reason, we first cloned and characterized the response of several key genes for the production and action of testicular steroids to the in vivo administration of human chorionic gonadotropin (hCG) and, second, we investigated the transcriptomic effects of hCG in the Senegalese sole testis. We succeeded in cloning the full-length cDNAs for Steroidogenic Acute Regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-HSD and 20β-HSD and a partial cDNA for the nuclear progesterone receptor. In this study we also identified a transcript encoding a protein with homology to StAR, which we named StAR-like, that could represent a new member of the StAR-related lipid transfer (START) family. All the cloned genes were expressed in the testis and their expression levels were significantly increased by the in vivo administration of hCG. The plasma levels of testosterone and 11-ketotestosterone also increased in response to hCG administration, likely as a result of the induction of the expression of steroidogenic enzymes by hCG. Furthermore, gene expression analysis by microarray identified 90 differentially expressed genes in the testis in response to hCG administration, including genes potentially involved in steroidogenesis, progression of spermatogenesis and germ cell maturation and cytoskeletal organization. Our results have identified for the first time a number of key genes involved in the regulation of steroid production and spermatogenesis in the Senegalese sole testis that are under gonadotropic control.

Hyperinsulinemia induces insulin resistance and immune suppression via Ptpn6/Shp1 in zebrafish

Type 2 diabetes, obesity, and metabolic syndrome are pathologies where insulin resistance plays a central role, and that affect a large population worldwide. These pathologies are usually associated with a dysregulation of insulin secretion leading to a chronic exposure of the tissues to high insulin levels (i.e. hyperinsulinemia), which diminishes the concentration of key downstream elements, causing insulin resistance. The complexity of the study of insulin resistance arises from the heterogeneity of the metabolic states where it is observed. To contribute to the understanding of the mechanisms triggering insulin resistance, we have developed a zebrafish model to study insulin metabolism and its associated disorders. Zebrafish larvae appeared to be sensitive to human recombinant insulin, becoming insulin-resistant when exposed to a high dose of the hormone. Moreover RNA-seq-based transcriptomic profiling of these larvae revealed a strong downregulation of a number of immune-relevant genes as a consequence of the exposure to hyperinsulinemia. Interestingly, as an exception, the negative immune modulator protein tyrosine phosphatase nonreceptor type 6 (ptpn6) appeared to be upregulated in insulin-resistant larvae. Knockdown of ptpn6 was found to counteract the observed downregulation of the immune system and insulin signaling pathway caused by hyperinsulinemia. These results indicate that ptpn6 is a mediator of the metabolic switch between insulin-sensitive and insulin-resistant states. Our zebrafish model for hyperinsulinemia has therefore demonstrated its suitability for discovery of novel regulators of insulin resistance. In addition, our data will be very useful in further studies of the function of immunological determinants in a non-obese model system.

Mechanisms Regulating GLUT4 Transcription in Skeletal Muscle Cells Are Highly Conserved across Vertebrates

The glucose transporter 4 (GLUT4) plays a key role in glucose uptake in insulin target tissues. This transporter has been extensively studied in many species in terms of its function, expression and cellular traffic and complex mechanisms are involved in its regulation at many different levels. However, studies investigating the transcription of the GLUT4 gene and its regulation are scarce. In this study, we have identified the GLUT4 gene in a teleost fish, the Fugu (Takifugu rubripes), and have cloned and characterized a functional promoter of this gene for the first time in a non-mammalian vertebrate. In silico analysis of the Fugu GLUT4 promoter identified potential binding sites for transcription factors such as SP1, C/EBP, MEF2, KLF, SREBP-1c and GC-boxes, as well as a CpG island, but failed to identify a TATA box. In vitro analysis revealed three transcription start sites, with the main residing 307 bp upstream of the ATG codon. Deletion analysis determined that the core promoter was located between nucleotides -132/+94. By transfecting a variety of 5´deletion constructs into L6 muscle cells we have determined that Fugu GLUT4 promoter transcription is regulated by insulin, PG-J2, a PPARγ agonist, and electrical pulse stimulation. Furthermore, our results suggest the implication of motifs such as PPARγ/RXR and HIF-1α in the regulation of Fugu GLUT4 promoter activity by PPARγ and contractile activity, respectively. These data suggest that the characteristics and regulation of the GLUT4 promoter have been remarkably conserved during the evolution from fish to mammals, further evidencing the important role of GLUT4 in metabolic regulation in vertebrates.

Stage-specific gene expression during spermatogenesis in the Senegalese sole (Solea senegalensis), a fish with semi-cystic type of spermatogenesis, as assessed by laser capture microdissection and absolute quantitative PCR

Spermatogenesis is a complex process where hormonal signals regulate the interaction of different cell types in a tight spatial and temporal fashion. The Senegalese sole (Solea senegalensis) is a marine flatfish that, in contrast to many fish, exhibits a semi-cystic, asynchronous pattern of spermatogenesis progression. This pattern is characterized by the release of spermatids into the tubule lumen, where they transform into spermatozoa. In this study, we used laser capture microdissection (LCM) to isolate cells from cysts containing spermatogonia, spermatocytes, spermatids or spermatozoa in order to investigate developmental patterns of gene expression. Furthermore, we also analyzed the stage-specific expression of the same set of genes throughout spermatogenesis (early-mid, late and maturing spermatogenic stages) in tissue fragments of the Senegalese sole testis. Genes analyzed by absolute qPCR in cysts isolated by LCM and stage-specific testis samples included genes involved in steroid synthesis and action (3β-hsd, 17β-hsd, 20β-hsd, star, star-like, progesterone receptor), gonadotropin action (fshr, lhr), the kisspeptin system (kiss2, kiss2r) and other genes important for the production of mature gametes (zona pellucida 2.2, claudin and clusterin). Our results show that, in general, steroidogenesis-related genes tended to increase with spermatogenesis progression and that 3β-hsd and 20β-hsd were expressed in germ cells but 17β-hsd was not. Our results also show that fshr is expressed in most testicular cell types, including germ cells. In contrast, lhr is expressed only in late spermatogenesis and is not expressed in any of the germ cell types examined, indicating that, in contrast to fshr, lhr may be primarily expressed in non-germinal cells (e.g. Leydig cells). Furthermore, kisspeptin and its receptor were expressed in all germ cell types examined and, as expected, gamete maturation-related genes were more expressed in mature stages. These results illustrate that key factors that participate in the hormonal regulation of spermatogenesis in the Senegalese sole testis show complex cell type- and stage-specific patterns of gene expression.

Establishment and optimization of a high throughput setup to study Staphylococcus epidermidis and Mycobacterium marinum infection as a model for drug discovery.

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

GLUT2-mediated glucose uptake and availability are required for embryonic brain development in zebrafish

Glucose transporter 2 (GLUT2; gene name SLC2A2) has a key role in the regulation of glucose dynamics in organs central to metabolism. Although GLUT2 has been studied in the context of its participation in peripheral and central glucose sensing, its role in the brain is not well understood. To decipher the role of GLUT2 in brain development, we knocked down slc2a2 (glut2), the functional ortholog of human GLUT2, in zebrafish. Abrogation of glut2 led to defective brain organogenesis, reduced glucose uptake and increased programmed cell death in the brain. Coinciding with the observed localization of glut2 expression in the zebrafish hindbrain, glut2 deficiency affected the development of neural progenitor cells expressing the proneural genes atoh1b and ptf1a but not those expressing neurod. Specificity of the morphant phenotype was demonstrated by the restoration of brain organogenesis, whole-embryo glucose uptake, brain apoptosis, and expression of proneural markers in rescue experiments. These results indicate that glut2 has an essential role during brain development by facilitating the uptake and availability of glucose and support the involvement of glut2 in brain glucose sensing.

Structural and Functional Evolution of Glucose Transporter 4 (GLUT4): A Look at GLUT4 in Fish

The insulin-responsive glucose transporter GLUT4 was first described in 1988 as a result of studies on the regulation of glucose metabolism by insulin [1]. Soon after the discovery of GLUT4, several groups cloned GLUT4 in the human [2], rat [3,4] and mouse [5]. Since its discovery, GLUT4 has received, together with GLUT1, more experimental attention than any other single membrane transport protein. Structurally, GLUT4 follows the predicted model for class I glucose transporters. GLUT4 has a high affinity for glucose, with a Km of approximately 5 mM [6], and also transports mannose, galactose, dehydroascorbic acid and glucosamine [7-10]. In mammals, GLUT4 is mainly expressed in cardiac and skeletal muscle, brown and white adipose tissue, and brain [6,11,12]. GLUT4 plays a pivotal role in whole body glucose homeostasis, mediating the uptake of glucose regulated by insulin [13,14]. GLUT4 is responsible for the reduction in the postprandial rise in plasma glucose levels [6]. Insulin acts by stimulating the translocation of specific GLUT4-containing vesicles from intracellular stores to the plasma membrane (PM) resulting in an immediate increase in glucose transport [6,15]. The disruption of GLUT4 expression has been extensively associat‐ ed with pathologies of impaired glucose uptake and insulin resistance such as type 2 diabetes and obesity [13,16-18].

Transcriptional regulation of the gilthead seabream (Sparus aurata) interleukin-6 gene promoter

Interleukin-6 (IL-6) has been identified and characterized from several fish species and its mRNA expression is induced by pathogen-associated molecular patterns (PAMPs) and cytokines in immune cells and tissues. However, the transcriptional regulation of the IL-6 gene in fish is not well understood. In the present study, we have cloned and sequenced a 1028 bp 5-flanking DNA region from the IL-6 gene in seabream (Sparus aurata). Sequence analysis of the seabream IL-6 promoter (sbIL-6P) evidenced the presence of a conserved TATA motif and conserved response elements for NF-κB, C/EBPβ (NF-IL6), AP-1 and GRE, similar to other vertebrate IL-6 promoters. Functional characterization of sbIL-6P was performed by cloning sbIL-6P into a luciferase expression vector and by transfecting it into L6 muscle cells, a mammalian cell line shown previously to express IL-6 in response to pro-inflammatory stimuli. We show here that the activity of sbIL-6P was significantly induced by pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα), IL-6 and IL-2, as well as by lipopolysaccharide (LPS), but significantly repressed by dexamethasone. In addition, the stimulatory effects of TNFα on sbIL-6P activity appeared to be mediated by the NF-κB, p38 MAPK and JNK signaling pathways. Deletion analyses of sbIL-6P suggested that activation of sbIL-6P by TNFα and IL-6 required the presence of binding motifs present in the proximal promoter (-171 to -84) whereas activation by IL-2 required binding motifs present in the distal promoter (-1024 to -864). The results from this study indicate, for the first time in fish, that pro-inflammatory cytokines, LPS and glucocorticoids can regulate the activity of the IL-6 gene at a transcriptional level and identify important regions in its response to cytokines.