Zebrafish Models in NeuroPsychopharmacology and CNS Drug Discovery

Despite the high prevalence of neuropsychiatric disorders, their aetiology and molecular mechanisms remain poorly understood. The zebrafish (Danio rerio) is increasingly utilized as a powerful animal model in neuropharmacology research and in vivo drug screening. Collectively, this makes zebrafish a useful tool for drug discovery and the identification of disordered molecular pathways. Here, we discuss zebrafish models of selected human neuropsychiatric disorders and drug-induced phenotypes. As well as covering a broad range of brain disorders (from anxiety and psychoses to neurodegeneration), we also summarize recent developments in zebrafish genetics and small molecule screening, which markedly enhance the disease modelling and the discovery of novel drug targets

Effect of MC4R gene polymorphism on food intake in adolescents with overweight and obesity

BACKGROUND: The melanocortin 4 receptor gene (MC4R) codes the receptor expressed in the hypothalamus and involved in the regulation of body mass and height. Data on the association of polymorphism MC4R rs17782313 with anthropometric parameters are contradictory. AIMS: to study the influence of the carrier of polymorphism MC4R rs17782313 on the anthropometric parameters in adolescents of different ethnic groups: caucasians and mongoloids living in the Siberia. MATERIALS AND METHODS: The study included 179 caucasian adolescents (by the example of russians, average age is 15.07 ± 1.25 years) and 182 mongoloid adolescents (by the example of the buryats, the average age is 14.71 ± 1.28 years), 89 and 92 adolescents were included in groups with overweight and obesity (standard deviation (SDS) BMI> 1), in the control groups (SDS BMI from -1 to + 1) also 90 and 90 adolescents were included in the control groups (SDS BMI from -1 to + 1) (russian and buryat, respectively). Anthropometric measurements included height, weight with the calculation of BMI and SDS BMI, WC (waist circumference), HC (hip circumference ). Genotyping was performed by real-time PCR. Statistical analysis of the results of the study was carried out using the software «STATISTICA 8.0». RESULTS: We showed no association of the risky C-allele of polymorphism rs17782313 with overweight and obesity in russian adolescents (22.5% vs 17.9% OR = 1.34 (p> 0.05)) and in the buryat (29.8% vs 24.1%, OR = 1.43 (p> 0.05)). It was revealed that adolescent carriers of the C-allele in buryat showed higher growth in both groups (control: 162.19 cm vs 157.26 cm (p = 0.019)), the main group: 165.24 cm vs 164.91 cm (p = 0.041)), as well as weight gain in the control group (52.29 kg vs 48.05 kg (p = 0.028)). CONCLUSIONS: Thus, the study revealed the relationship of MC4R rs17782313 polymorphism with height and weight in adolescents of buryat ethnic group

Late Replication Domains in Polytene and Non-Polytene Cells of Drosophila melanogaster

In D. melanogaster polytene chromosomes, intercalary heterochromatin (IH) appears as large dense bands scattered in euchromatin and comprises clusters of repressed genes. IH displays distinctly low gene density, indicative of their particular regulation. Genes embedded in IH replicate late in the S phase and become underreplicated. We asked whether localization and organization of these late-replicating domains is conserved in a distinct cell type. Using published comprehensive genome-wide chromatin annotation datasets (modENCODE and others), we compared IH organization in salivary gland cells and in a Kc cell line. We first established the borders of 60 IH regions on a molecular map, these regions containing underreplicated material and encompassing ∼12% of Drosophila genome. We showed that in Kc cells repressed chromatin constituted 97% of the sequences that corresponded to IH bands. This chromatin is depleted for ORC-2 binding and largely replicates late. Differences in replication timing between the cell types analyzed are local and affect only sub-regions but never whole IH bands. As a rule such differentially replicating sub-regions display open chromatin organization, which apparently results from cell-type specific gene expression of underlying genes. We conclude that repressed chromatin organization of IH is generally conserved in polytene and non-polytene cells. Yet, IH domains do not function as transcription- and replication-regulatory units, because differences in transcription and replication between cell types are not domain-wide, rather they are restricted to small “islands” embedded in these domains. IH regions can thus be defined as a special class of domains with low gene density, which have narrow temporal expression patterns, and so displaying relatively conserved organization

Underreplicated regions in Drosophila melanogaster are enriched with fast-evolving genes and highly conserved noncoding sequences

Many late replicating regions are underreplicated in polytene chromosomes of Drosophila melanogaster. These regions contain silenced chromatin and overlap long syntenic blocks of conserved gene order in drosophilids. In this report we show that in D. melanogaster the underreplicated regions are enriched with fast-evolving genes lacking homologs in distant species such as mosquito or human, indicating that the phylogenetic conservation of genes correlates with replication timing and chromatin status. Drosophila genes without human homologs located in the underreplicated regions have higher nonsynonymous substitution rate and tend to encode shorter proteinswhen compared with those in the adjacent regions. At the same time, the underreplicated regions are enriched with ultraconserved elements and highly conserved noncoding sequences, especially in introns of very long genes indicating the presence of an extensive regulatory network that may be responsible for the conservation of gene order in these regions. The regions have amodest preference for long noncoding RNAs but are depleted for small nucleolar RNAs, microRNAs, and transfer RNAs. Our results demonstrate that the underreplicated regions have a specific genic composition and distinct pattern of evolution

Similarity in replication timing between polytene and diploid cells is associated with the organization of the <i>Drosophila</i> genome

<div><p>Morphologically, polytene chromosomes of <i>Drosophila melanogaster</i> consist of compact “black” bands alternating with less compact “grey” bands and interbands. We developed a comprehensive approach that combines cytological mapping data of FlyBase-annotated genes and novel tools for predicting cytogenetic features of chromosomes on the basis of their protein composition and determined the genomic coordinates for all black bands of polytene chromosome 2R. By a PCNA immunostaining assay, we obtained the replication timetable for all the bands mapped. The results allowed us to compare replication timing between polytene chromosomes in salivary glands and chromosomes from cultured diploid cell lines and to observe a substantial similarity in the global replication patterns at the band resolution level. In both kinds of chromosomes, the intervals between black bands correspond to early replication initiation zones. Black bands are depleted of replication initiation events and are characterized by a gradient of replication timing; therefore, the time of replication completion correlates with the band length. The bands are characterized by low gene density, contain predominantly tissue-specific genes, and are represented by silent chromatin types in various tissues. The borders of black bands correspond well to the borders of topological domains as well as to the borders of the zones showing H3K27me3, SUUR, and LAMIN enrichment. In conclusion, the characteristic pattern of polytene chromosomes reflects partitioning of the <i>Drosophila</i> genome into two global types of domains with contrasting properties. This partitioning is conserved in different tissues and determines replication timing in <i>Drosophila</i>.</p></div

All rb-bands display delayed replication in salivary gland polytene chromosomes and correspond to local minima on replication profiles for cell cultures.

<p><b>(A)</b> The replication pattern in salivary gland polytene chromosomes with all rb-bands labeled by PCNA in the region 43F-46B of polytene chromosome 2R as an example. The very existence of a pattern like this suggests that rb-bands are replicated later than the intervals between them. <b>(B, C)</b> Locations of rb-bands scaled to the genome map (B) and replication profiles in the cell cultures in comparison: data on KC, Cl8, S2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref033" target="_blank">33</a>], and BG3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref050" target="_blank">50</a>] (C) suggest that local minima on replication profiles for the cell cultures fall within rb-bands. This in turn suggests that rb-bands replicate later than their flanking regions. <b>(D)</b> Distribution of ORC2 peaks in Kc cells and in salivary glands [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref051" target="_blank">51</a>] shows typical zones of low peak density in rb-bands and a prominent ORC2 signal confined to the regions between the rb-bands.</p

The logic of changing patterns in polytene chromosomes.

<p><b>Links between these patterns and events at the DNA sequence level. (A)</b> A schematic of replication fork locations and the corresponding PCNA patterns in the zone of alternating grey bands and interbands and in an rb-band (black in the figure). <b>(B)</b> A schematic of a polytene chromosome region with three rb-bands (black in the figure) of different lengths. Intervals between rb-bands appear as grey bands and interbands. <b>(C)</b> Consecutive changes in PCNA binding patterns (red in the figure) during the S phase. Top to bottom: continuous labeling substage; the substage at which the label is seen in all rb-bands; the late S phase, when only the thickest bands get labeled. The central part of the thickest band contains a region that never undergoes replication. <b>(D)</b> Replication fork locations depending on time (time passes in the downward direction). Replication fork locations were inferred under the assumption that replication is initiated randomly at each INT point once per INT in each replication cycle, with little asynchrony. Differences in replication rates between different genomic regions were ignored. The portion of the profile corresponding to the under-replication zone is beyond the S phase. If the differences in replication rates between rb-bands were to be considered, the local minima would be deeper. <b>(E)</b> A model reflecting similarities and differences in replication timing between diploid cell chromosomes and salivary gland polytene chromosomes. Averaged replication profiles are identical in early replication initiation zones. A portion of the genome in salivary gland polytene chromosomes is slower to replicate because of slow replication in “black” bands and the absence of late origin firing; this portion stays under-replicated.</p

Localization of polytene chromosomes’ black bands on the Drosophila genome map could be predicted by means of data on the distribution of interband-specific chromatin proteins in cell cultures: Region 43F-46B of polytene chromosome 2R as an example.

<p><b>(A)</b> Distribution of four chromatin types [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref004" target="_blank">4</a>]. Ruby chromatin (depicted in magenta) is devoid of active chromatin markers. Aquamarine chromatin (depicted in cyan) is enriched with all the proteins that are typical of polytene chromosome interbands (see text). <b>(B)</b> Locations of condensed bands in polytene chromosomes as predicted from the distribution of ruby chromatin and aquamarine chromatin (each of these bands appears as an interval between aquamarine segments that include ruby chromatin) (see A). <b>(C)</b> A distribution of nine chromatin states in S2 cells (top) and Bg3 cells (bottom) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref022" target="_blank">22</a>]. The red color corresponds to chromatin type enriched with active promoters. The presence of this chromatin type in both cell cultures at once is one of the markers of polytene chromosome interbands. <b>(D)</b> Enrichment peaks of CHRIZ, the most typical interband protein, in the chromosomes of four cell lines (top to bottom: Bg3, Kc, S2, and Cl8) (modENCODE data). <b>(E)</b> Localization of the predicted positions of compacted bands within the framework of the 4-state model combined with the interband criterion filtering (see C, D). <b>(F)</b> FlyBase CytoMap locations of compacted bands corresponding to rb-bands (see G) (<a href="http://flybase.org/" target="_blank">http://flybase.org</a>). <b>(G)</b> Localization of the predicted positions of compacted bands after all correction steps (rb-bands, see text). Bands were assigned names according to mapping data in FlyBase (see text) and Bridges’ detailed map ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref047" target="_blank">47</a>], see J). <b>(H–J)</b> Locations of compacted bands in polytene chromosomes from salivary glands (H, J) and pseudonurse cells of <i>otu</i>^<i>11</i> mutants (H, reprinted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref011" target="_blank">11</a>] under a CC BY license, with permission from Springer Nature: Chromosome Research, original copyright [1995]) in comparison and their correspondence to the bands predicted by the analysis of protein distribution in the cell cultures (G). Compacted black bands as visualized by aceto-orcein staining (H, I) and according to Bridges’ detailed map [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref047" target="_blank">47</a>] (J).</p

A flow diagram showing the main steps of mapping of black bands.

<p>A flow diagram showing the main steps of mapping of black bands.</p

Cytological mapping of the binding sites of anti-PCNA antibodies breaks down the S phase of the endocycle into six substages and assigns rb-bands to replication completion groups.

<p><b>First column:</b> The replication pattern of bands in the regions 43F-46B of polytene chromosome 2R as visualized by antibodies against PCNA (red) on <i>SuUR</i>^<i>ES</i> (A) and Oregon R (B–F) polytene chromosomes. Top to bottom: consecutive substages of the S phase, from the earliest (A) to latest (F). (A) “Black” bands not labeled yet. (B–F) Discontinuous labeling. Band designation appears at the substages at which these bands end replication. <b>Second column:</b> superimposed images, immunolocalization and phase contrast. <b>Third column:</b> Replication pattern-based substages of S phase, where ER is the earliest substage and LR1–LR5 are the subsequent provisionally late substages. <b>Fourth column:</b> Each rb-band was assigned to one of five groups, LR1 through LR5, according to the substage at which this rb-band ends replication. A small group of bands with no PCNA at LR1 was classified as LR0. For each substage, all replicating groups and all bands that completed replication are indicated (red text).</p