Genetics in Drug Discovery.

Drug discovery is a complex process with high attrition rate: only about half of the compounds in advanced preclinical stages actually enter human trials. Key to these failures is our lack of understanding of human biology and the difficulties in translating our preclinical knowledge into cures. Here, we examine how genetics can be leveraged in drug discovery to understand and alter human biology.

ALKALs are in vivo ligands for ALK family receptor tyrosine kinases in the neural crest and derived cells.

Mutations in anaplastic lymphoma kinase (ALK) are implicated in somatic and familial neuroblastoma, a pediatric tumor of neural crest-derived tissues. Recently, biochemical analyses have identified secreted small ALKAL proteins (FAM150, AUG) as potential ligands for human ALK and the related leukocyte tyrosine kinase (LTK). In the zebrafish Danio rerio, DrLtk, which is similar to human ALK in sequence and domain structure, controls the development of iridophores, neural crest-derived pigment cells. Hence, the zebrafish system allows studying Alk/Ltk and Alkals involvement in neural crest regulation in vivo. Using zebrafish pigment pattern formation, Drosophila eye patterning, and cell culture-based assays, we show that zebrafish Alkals potently activate zebrafish Ltk and human ALK driving downstream signaling events. Overexpression of the three DrAlkals cause ectopic iridophore development, whereas loss-of-function alleles lead to spatially distinct patterns of iridophore loss in zebrafish larvae and adults. alkal loss-of-function triple mutants completely lack iridophores and are larval lethal as is the case for ltk null mutants. Our results provide in vivo evidence of (i) activation of ALK/LTK family receptors by ALKALs and (ii) an involvement of these ligand-receptor complexes in neural crest development.

Correction: Sensory Neuron-Derived Eph Regulates Glomerular Arbors and Modulatory Function of a Central Serotonergic Neuron.

[This corrects the article DOI: 10.1371/journal.pgen.1003452.].

How fish color their skin: A paradigm for development and evolution of adult patterns: Multipotency, plasticity, and cell competition regulate proliferation and spreading of pigment cells in Zebrafish coloration.

Pigment cells in zebrafish - melanophores, iridophores, and xanthophores - originate from neural crest-derived stem cells associated with the dorsal root ganglia of the peripheral nervous system. Clonal analysis indicates that these progenitors remain multipotent and plastic beyond embryogenesis well into metamorphosis, when the adult color pattern develops. Pigment cells share a lineage with neuronal cells of the peripheral nervous system; progenitors propagate along the spinal nerves. The proliferation of pigment cells is regulated by competitive interactions among cells of the same type. An even spacing involves collective migration and contact inhibition of locomotion of the three cell types distributed in superimposed monolayers in the skin. This mode of coloring the skin is probably common to fish, whereas different patterns emerge by species specific cell interactions among the different pigment cell types. These interactions are mediated by channels involved in direct cell contact between the pigment cells, as well as unknown cues provided by the tissue environment.

Analysis of the SWI/SNF chromatin-remodeling complex during early heart development and BAF250a repression cardiac gene transcription during P19 cell differentiation.

The regulatory networks of differentiation programs and the molecular mechanisms of lineage-specific gene regulation in mammalian embryos remain only partially defined. We document differential expression and temporal switching of BRG1-associated factor (BAF) subunits, core pluripotency factors and cardiac-specific genes during post-implantation development and subsequent early organogenesis. Using affinity purification of BRG1 ATPase coupled to mass spectrometry, we characterized the cardiac-enriched remodeling complexes present in E8.5 mouse embryos. The relative abundance and combinatorial assembly of the BAF subunits provides functional specificity to Switch/Sucrose NonFermentable (SWI/SNF) complexes resulting in a unique gene expression profile in the developing heart. Remarkably, the specific depletion of the BAF250a subunit demonstrated differential effects on cardiac-specific gene expression and resulted in arrhythmic contracting cardiomyocytes in vitro. Indeed, the BAF250a physically interacts and functionally cooperates with Nucleosome Remodeling and Histone Deacetylase (NURD) complex subunits to repressively regulate chromatin structure of the cardiac genes by switching open and poised chromatin marks associated with active and repressed gene expression. Finally, BAF250a expression modulates BRG1 occupancy at the loci of cardiac genes regulatory regions in P19 cell differentiation. These findings reveal specialized and novel cardiac-enriched SWI/SNF chromatin-remodeling complexes, which are required for heart formation and critical for cardiac gene expression regulation at the early stages of heart development.

Sensory neuron-derived eph regulates glomerular arbors and modulatory function of a central serotonergic neuron.

Olfactory sensory neurons connect to the antennal lobe of the fly to create the primary units for processing odor cues, the glomeruli. Unique amongst antennal-lobe neurons is an identified wide-field serotonergic neuron, the contralaterally-projecting, serotonin-immunoreactive deutocerebral neuron (CSDn). The CSDn spreads its termini all over the contralateral antennal lobe, suggesting a diffuse neuromodulatory role. A closer examination, however, reveals a restricted pattern of the CSDn arborization in some glomeruli. We show that sensory neuron-derived Eph interacts with Ephrin in the CSDn, to regulate these arborizations. Behavioural analysis of animals with altered Eph-ephrin signaling and with consequent arborization defects suggests that neuromodulation requires local glomerular-specific patterning of the CSDn termini. Our results show the importance of developmental regulation of terminal arborization of even the diffuse modulatory neurons to allow them to route sensory-inputs according to the behavioural contexts.

Dendritic refinement of an identified neuron in the Drosophila CNS is regulated by neuronal activity and Wnt signaling.

The dendrites of neurons undergo dramatic reorganization in response to developmental and other cues, such as stress and hormones. Although their morphogenesis is an active area of research, there are few neuron preparations that allow the mechanistic study of how dendritic fields are established in central neurons. Dendritic refinement is a key final step of neuronal circuit formation and is closely linked to emergence of function. Here, we study a central serotonergic neuron in the Drosophila brain, the dendrites of which undergo a dramatic morphological change during metamorphosis. Using tools to manipulate gene expression in this neuron, we examine the refinement of dendrites during pupal life. We show that the final pattern emerges after an initial growth phase, in which the dendrites function as 'detectors', sensing inputs received by the cell. Consistent with this, reducing excitability of the cell through hyperpolarization by expression of K(ir)2.1 results in increased dendritic length. We show that sensory input, possibly acting through NMDA receptors, is necessary for dendritic refinement. Our results indicate that activity triggers Wnt signaling, which plays a 'pro-retraction' role in sculpting the dendritic field: in the absence of sensory input, dendritic arbors do not retract, a phenotype that can be rescued by activating Wnt signaling. Our findings integrate sensory activity, NMDA receptors and Wingless/Wnt5 signaling pathways to advance our understanding of how dendritic refinement is established. We show how the maturation of sensory function interacts with broadly distributed signaling molecules, resulting in their localized action in the refinement of dendritic arbors.

SOX8 regulates permeability of the blood-testes barrier that affects adult male fertility in the mouse.

Sertoli cells provide nutritional and physical support to germ cells during spermatogenesis. Sox8 encodes a member of the high mobility group of transcription factors closely related to Sox9 and Sox10. Sertoli cells express SOX8 protein, and its elimination results in an age-dependent dysregulation of spermatogenesis, causing adult male infertility. Among the claudin genes with altered expression in the Sox8(-/-) testes, was claudin-3, which is required for the regulation and maintenance of the blood-testes barrier (BTB). Because the BTB is critical in restricting small molecules in the luminal compartment of the seminiferous tubules, the aim of this study was to analyze the level of tight junction proteins (claudin-3, claudin-11, and occludin) and BTB permeability in Sox8(-/-) adult testes. The acetylation level of alpha-tubulin and microtubule organization was also evaluated because microtubules are critical in maintaining the microenvironment of the seminiferous epithelium. Western blot analysis shows that claudin-3 protein is decreased in Sox8(-/-) testes. Chromatin immunoprecipitation confirmed that SOX8 binds at the promoter region of claudin-3. Claudin-3 was localized to the Sertoli cell tight junctions of wild-type testes and significantly decreased in the Sox8(-/-) testes. The use of biotin tracers showed increased BTB permeability in the Sox8(-/-) adult testes. Electron microscopy analysis showed that microtubule structures were destabilized in the Sox8(-/-) testes. These results suggest that Sox8 is essential in Sertoli cells for germ cell differentiation, partly by controlling the microenvironment of the seminiferous epithelium.

Bmpr1a is required for proper migration of the AVE through regulation of Dkk1 expression in the pre-streak mouse embryo.

Here, we report a novel mechanism regulating migration of the anterior visceral endoderm (AVE) by BMP signaling through BMPRIA. In Bmpr1a-deficient (Bmpr-null) embryos, the AVE does not migrate at all. In embryos with an epiblast-specific deletion of Bmpr1a (Bmpr1a(null/flox); Sox2Cre embryos), the AVE cells migrate randomly from the distal end of embryos, resulting in an expansion of the AVE. Dkk1, which is normally expressed in the anterior proximal visceral endoderm (PxVE), is downregulated in Bmpr-null embryos, whereas it is circumferentially expressed in Bmpr1a(null/flox); Sox2Cre embryos at E5.75-6.5. These results demonstrate an association of the position of Dkk1 expressing cells with direction of the migration of AVE. In Bmpr1a(null/flox); Sox2Cre embryos, a drastic decrease of WNT signaling is observed at E6.0. Addition of WNT3A to the culture of Bmpr1a(null/flox); Sox2Cre embryos at E5.5 restores expression patterns of Dkk1 and Cer1. These data indicate that BMP signaling in the epiblast induces Wnt3 and Wnt3a expression to maintain WNT signaling in the VE, resulting in downregulation of Dkk1 to establish the anterior expression domain. Thus, our results suggest that BMP signaling regulates the expression patterns of Dkk1 for anterior migration of the AVE.

αKlotho Regulates Age-Associated Vascular Calcification and Lifespan in Zebrafish.

The hormone αKlotho regulates lifespan in mice, as knockouts die early of what appears to be accelerated aging due to hyperphosphatemia and soft tissue calcification. In contrast, the overexpression of αKlotho increases lifespan. Given the severe mouse phenotype, we generated zebrafish mutants for αklotho as well as its binding partner fibroblast growth factor-23 (fgf23). Both mutations cause shortened lifespan in zebrafish, with abrupt onset of behavioral and degenerative physical changes at around 5 months of age. There is a calcification of vessels throughout the body, most dramatically in the outflow tract of the heart, the bulbus arteriosus (BA). This calcification is associated with an ectopic activation of osteoclast differentiation pathways. These findings suggest that the gradual loss of αKlotho found in normal aging might give rise to ectopic calcification.

Multiple alternative splicing of Dmrt1 during gonadogenesis in Indian mugger, a species exhibiting temperature-dependent sex determination.

Dmrt1 is an evolutionarily conserved gene having important role in the sex determination from lower vertebrates to mammals. Recent studies show transcriptional diversity for this important gene during gonadal differentiation in a few vertebrate species having genetic sex determination (GSD). In this study, we show for the first time that the transcriptional diversity of Dmrt1 is also found in the Indian mugger that exhibits temperature-dependent sex determination (TSD). We report here isolation and characterization of eight novel isoforms of Dmrt1 from Crocodylus palustris, along with its genomic locus that is referred as, cpDmrt1. Further, by sequence comparisons of cpDmrt1 and its expressed isoforms, we demonstrate that all the isoforms are generated by alternative splicing, exonization of intronic sequences and alternative polyA sites from the same locus. The eight transcripts range from 494 to 2060 bp and encode six predicted proteins having the characteristic DM domain of Dmrt1. The major heterogeneity in the isoforms and their predicted proteins is seen only in their C-termini and 3'-UTRs, which do not match with any similar sequences reported for other vertebrates. The cpDmrt1 expression was seen mainly in developing GAM (genital ridge-adrenal-mesonephros complex) with significant upregulation only in male embryos from the start of the temperature sensitive period (TSP). More significantly, approximately 70% of this expression was contributed by only one isoform (cpDmrt1e) that also has a unique 15 amino acid domain towards its C-terminal. cpDmrt1 expression was also detected at a lower level in brain and developing kidney. The study thus provides the first account of Dmrt1 locus, its transcriptional diversity and sex-specific expression in Indian mugger, a TSD species.

A role for BRG1 in the regulation of genes required for development of the lymphatic system.

Lymphatic vasculature is an important part of the cardiovascular system with multiple functions, including regulation of the return of interstitial fluid (lymph) to the bloodstream, immune responses, and fat absorption. Consequently, lymphatic vasculature defects are involved in many pathological processes, including tumor metastasis and lymphedema. BRG1 is an important player in the developmental window when the lymphatic system is initiated. In the current study, we used tamoxifen inducible Rosa26CreERT2-BRG1floxed/floxed mice that allowed temporal analysis of the impact of BRG1 inactivation in the embryo. The BRG1floxed/floxed/Cre-TM embryos exhibited edema and hemorrhage at embryonic day-13 and began to die. BRG1 deficient embryos had abnormal lymphatic sac linings with fewer LYVE1 positive lymphatic endothelial cells. Indeed, loss of BRG1 attenuated expression of a subset of lymphatic genes in-vivo. Furthermore, BRG1 binds at the promoters of COUP-TFII and LYVE1, suggesting that BRG1 modulates expression of these genes in the developing embryos. Conversely, re-expression of BRG1 in cells lacking endogenous BRG1 resulted in induction of lymphatic gene expression in-vitro, suggesting that BRG1 was both required and sufficient for lymphatic gene expression. These studies provide important insights into intrinsic regulation of BRG1-mediated lymphatic-gene expression, and further an understanding of lymphatic gene dysregulation in lymphedema and other disease conditions.

The Indian Magical Herb 'Sanjeevni' (Selaginella bryopteris L.) - A Promising Anti-inflammatory Phytomedicine for the Treatment of Patients with Inflammatory Skin Diseases.

OBJECTIVES: Selaginella bryopteris L. (family: Selaginaceae), is often used in traditional Indian systems of medicine for the prevention and cure of several disorders and for the treatment of patient with spermatorrhoea, venereal disease, constipation, colitis, urinary tract infections, fever, epilepsy, leucorrhoea, beri-beri and cancer. It is also used as a strength tonic. This study aimed to evaluate the mechanisms underlying the anti-inflammatory effects of topically administered aqueous, polar and non-polar methanolic fractions (10 mg/20 µL) of Selaginella bryopteris. METHODS: An acute oral toxicity study of Selaginella bryopteris at doses from 250 to 2,000 mg/kg body weight (bw) was performed. Aqueous, polar and non-polar methanolic extracts (10 mg/20 µL) applied topically for 5 days were evaluated for their anti-inflammatory effects against 12-tetra-O-decanoyl phorbol acetate (TPA)-induced inflammation by using the redness in the ear, the ear's weight (edema), oxidative stress parameters, such as lipid-peroxide (LPO) and nitric oxide (NO), and the pro-inflammatory cytokines involved in inflammation, such as tumour necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6. Indomethacine (0.5 mg/20 µL) was used for the positive control. RESULTS: Selaginella bryopteris produced no mortalities when administered orally at doses from 250 to 2,000 mg/kg bw. Topical treatment with the non-polar methanolic fraction (10 mg/20 αL) significantly suppressed redness (2.4 ± 0.5) and edema (30.4 ± 1) and effectively reduced the LPO level (32.3 ± 3.3). The NO level was (8.07 ± 0.55), and the TNF-α, IL-1ß, and IL-6 levels were decreased to 69.6 ± 15.5, 7.7 ± 4.8 and 82.6 ± 5.9, respectively. CONCLUSION: This study demonstrated for the first time the mechanisms underlying the anti-inflammatory effect of medicinal plants like Selaginella bryopteris and quantified the pharmacological interactions between them. The present study showed this herbal product to be a promising anti-inflammatory phytomedicine for the treatment of patients with inflammatory skin diseases.

The Developmental Genetics of Vertebrate Color Pattern Formation: Lessons from Zebrafish.

Color patterns are prominent features of many animals; they are highly variable and evolve rapidly leading to large diversities even within a single genus. As targets for natural as well as sexual selection, they are of high evolutionary significance. The zebrafish (Danio rerio) has become an important model organism for developmental biology and biomedical research in general, and it is the model organism to study color pattern formation in vertebrates. The fish display a conspicuous pattern of alternating blue and golden stripes on the body and on the anal and tail fins. This pattern is produced by three different types of pigment cells (chromatophores) arranged in precise layers in the hypodermis of the fish. In this essay, we will summarize the recent advances in understanding the developmental and genetic basis for stripe formation in the zebrafish. We will describe the cellular events leading to the formation of stripes during metamorphosis based on long-term lineage imaging. Mutant analysis has revealed that a number of signaling pathways are involved in the establishment and maintenance of the individual pigment cells. However, the striped pattern itself is generated by self-organizing mechanisms requiring interactions between all three pigment cell types. The involvement of integral membrane proteins, including connexins and potassium channels, suggests that direct physical contacts between chromatophores are involved, and that the directed transport of small molecules or bioelectrical coupling is important for these interactions. This mode of patterning by transmitting spatial information between adjacent tissues within three superimposed cell layers is unprecedented in other developmental systems. We propose that variations in the patterns among Danio species are caused by allelic differences in the genes responsible for these interactions.

High magnetic field induced otolith fusion in the zebrafish larvae.

Magnetoreception in animals illustrates the interaction of biological systems with the geomagnetic field (geoMF). However, there are few studies that identified the impact of high magnetic field (MF) exposure from Magnetic Resonance Imaging (MRI) scanners (>100,000 times of geoMF) on specific biological targets. Here, we investigated the effects of a 14 Tesla MRI scanner on zebrafish larvae. All zebrafish larvae aligned parallel to the B0 field, i.e. the static MF, in the MRI scanner. The two otoliths (ear stones) in the otic vesicles of zebrafish larvae older than 24 hours post fertilization (hpf) fused together after the high MF exposure as short as 2 hours, yielding a single-otolith phenotype with aberrant swimming behavior. The otolith fusion was blocked in zebrafish larvae under anesthesia or embedded in agarose. Hair cells may play an important role on the MF-induced otolith fusion. This work provided direct evidence to show that high MF interacts with the otic vesicle of zebrafish larvae and causes otolith fusion in an "all-or-none" manner. The MF-induced otolith fusion may facilitate the searching for MF sensors using genetically amenable vertebrate animal models, such as zebrafish.

Heterotypic interactions regulate cell shape and density during color pattern formation in zebrafish.

The conspicuous striped coloration of zebrafish is produced by cell-cell interactions among three different types of chromatophores: black melanophores, orange/yellow xanthophores and silvery/blue iridophores. During color pattern formation xanthophores undergo dramatic cell shape transitions and acquire different densities, leading to compact and orange xanthophores at high density in the light stripes, and stellate, faintly pigmented xanthophores at low density in the dark stripes. Here, we investigate the mechanistic basis of these cell behaviors in vivo, and show that local, heterotypic interactions with dense iridophores regulate xanthophore cell shape transition and density. Genetic analysis reveals a cell-autonomous requirement of gap junctions composed of Cx41.8 and Cx39.4 in xanthophores for their iridophore-dependent cell shape transition and increase in density in light-stripe regions. Initial melanophore-xanthophore interactions are independent of these gap junctions; however, subsequently they are also required to induce the acquisition of stellate shapes in xanthophores of the dark stripes. In summary, we conclude that, whereas homotypic interactions regulate xanthophore coverage in the skin, their cell shape transitions and density is regulated by gap junction-mediated, heterotypic interactions with iridophores and melanophores.

Zebrafish Leucocyte tyrosine kinase controls iridophore establishment, proliferation and survival.

The zebrafish striped pattern results from the interplay among three pigment cell types; black melanophores, yellow xanthophores and silvery iridophores, making it a valuable model to study pattern formation in vivo. It has been suggested that iridophore proliferation, dispersal and cell shape transitions play an important role during stripe formation; however, the underlying molecular mechanisms remain poorly understood. Using gain- and loss-of-function alleles of leucocyte tyrosine kinase (ltk) and a pharmacological inhibitor approach, we show that Ltk specifically regulates iridophore establishment, proliferation and survival. Mutants in shady/ltk lack iridophores and display an abnormal body stripe pattern. Moonstone mutants, ltk(mne) , display ectopic iridophores, suggesting hyperactivity of the mutant Ltk. The dominant ltk(mne) allele carries a missense mutation in a conserved position of the kinase domain that highly correlates with neuroblastomas in mammals. Chimeric analysis suggests a novel physiological role of Ltk in the regulation of iridophore proliferation by homotypic competition.

Homotypic cell competition regulates proliferation and tiling of zebrafish pigment cells during colour pattern formation.

The adult striped pattern of zebrafish is composed of melanophores, iridophores and xanthophores arranged in superimposed layers in the skin. Previous studies have revealed that the assembly of pigment cells into stripes involves heterotypic interactions between all three chromatophore types. Here we investigate the role of homotypic interactions between cells of the same chromatophore type. Introduction of labelled progenitors into mutants lacking the corresponding cell type allowed us to define the impact of competitive interactions via long-term in vivo imaging. In the absence of endogenous cells, transplanted iridophores and xanthophores show an increased rate of proliferation and spread as a coherent net into vacant space. By contrast, melanophores have a limited capacity to spread in the skin even in the absence of competing endogenous cells. Our study reveals a key role for homotypic competitive interactions in determining number, direction of migration and individual spacing of cells within chromatophore populations.

Pigment Cell Progenitors in Zebrafish Remain Multipotent through Metamorphosis.

The neural crest is a transient, multipotent embryonic cell population in vertebrates giving rise to diverse cell types in adults via intermediate progenitors. The in vivo cell-fate potential and lineage segregation of these postembryonic progenitors is poorly understood, and it is unknown if and when the progenitors become fate restricted. We investigate the fate restriction in the neural crest-derived stem cells and intermediate progenitors in zebrafish, which give rise to three distinct adult pigment cell types: melanophores, iridophores, and xanthophores. By inducing clones in sox10-expressing cells, we trace and quantitatively compare the pigment cell progenitors at four stages, from embryogenesis to metamorphosis. At all stages, a large fraction of the progenitors are multipotent. These multipotent progenitors have a high proliferation ability, which diminishes with fate restriction. We suggest that multipotency of the nerve-associated progenitors lasting into metamorphosis may have facilitated the evolution of adult-specific traits in vertebrates.

Zebrafish stripes as a model for vertebrate colour pattern formation.

Colour patterns are prominent features of many animals and have important functions in communication, such as camouflage, kin recognition and mate choice. As targets for natural as well as sexual selection, they are of high evolutionary significance. The molecular mechanisms underlying colour pattern formation in vertebrates are not well understood. Progress in transgenic tools, in vivo imaging and the availability of a large collection of mutants make the zebrafish (Danio rerio) an attractive model to study vertebrate colouration. Zebrafish display golden and blue horizontal stripes that form during metamorphosis as mosaics of yellow xanthophores, silvery or blue iridophores and black melanophores in the hypodermis. Lineage tracing revealed the origin of the adult pigment cells and their individual cellular behaviours during the formation of the striped pattern. Mutant analysis indicated that interactions between all three pigment cell types are required for the formation of the pattern, and a number of cell surface molecules and signalling systems have been identified as mediators of these interactions. The understanding of the mechanisms that underlie colour pattern formation is an important step towards deciphering the genetic basis of variation in evolution.