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Young Ninja Group (ages 3-5)

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Leonardo Watson
Leonardo Watson

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Turbot developmental stages selected during the metamorphosis process for transcriptome analysis. From left to right: (a) pre-metamorphic symmetrical larva prior to eye migration (stage 3b: 15dpf) (b) asymmetrical larva at the metamorphic climax with upper edge of migrating right eye visible from left side (stage 4d: 30dpf) (c) post-metamorphic asymmetrical juvenile with upper eye entirely placed on the left side (stage 5c: 57dpf). Fish were reared at 18 C. Scale bars (a) 2mm, (b,c) 5 mm.

CCC (Color Climax)


Visualization of gene expression datasets in turbot brains throughout the developmental metamorphosis process. MA plot of all transcriptome genes from pairwise comparisons: (a) pre-metamorphic vs. climax, (b) climax vs. post-metamorphic and (c) pre-metamorphic vs. post-metamorphic. The red dots plotted represent genes with an adjusted p-value d) Heatmap displaying the DEGs hierarchically clustered according to the expression profiles throughout metamorphosis. Each column represents an individual triplicate from each metamorphic stage (pre-metamorphic, climax and post-metamorphic) and each row represents different DEGs. The colors from light blue to dark blue indicate gene expression from low to high, respectively.

Main biological processes, molecular functions and cellular components boosted during metamorphosis in turbot brain. Gene Ontology (GO) enrichment of genes from hierarchical clustering showing high expression at (a) pre-metamorphic, (b) climax or (c) post-metamorphic stages. The most enriched GO terms (top 10, adjusted p-value

Selected clusters from Mfuzz soft clustering analysis of DEGs in turbot brains throughout the developmental metamorphosis process. DEGs show up-regulated genes corresponding to the: (a) pre-metamorphic stage, (b) climax stage, (c) post-metamorphic stage. Color code, from magenta to yellow, denote high or low Mfuzz membership values, respectively. Time 1, 2 and 3 in X-axis corresponds to pre-metamorphic, climax and post-metamorphic stage, respectively. (d) Validation of expression patterns of chit3, epd, thra, tshba, and ifih1 genes in the turbot brain using qPCR. In the bar plots, the trend lines represent the fold change obtained by analyzing the RNAseq values during the metamorphic stages. The bars represent the fold change values obtained by qPCR. Results were normalized to 18S gene and expressed as the mean SEM of two independent experiments. Data from climax stage was set at 1.

Main biological processes, molecular functions and cellular components boosted during metamorphosis in turbot brains. Gene Ontology (GO) enrichment of genes from Mfuzz clusters showing high expression at (a) pre-metamorphic, (b) climax or (c) post-metamorphic stages. The most enriched GO terms (top 10, adjusted p-value

The most significantly up-regulated and down-regulated DEGs in the turbot brain at the climax stage of metamorphosis. Venn diagrams show (a) up-regulated and (b) down-regulated DEGs after pairwise comparisons between metamorphic stages (pre-metamorphic vs. climax and climax vs. post-metamorphic). (c) Gene annotation proposed by Sma3s v2 software3.5, fold change values (Log2FC), p-values adjusted (padj) and read count of each sample.

Our results show a specific gene expression profile for each characterized developmental stage. In addition, we observed larger differences in the quantity of genes expressed in the pre-metamorphic stage with respect to the other stages, while the climax and post-metamorphosis stages presented a lower number of genes differentially expressed between them. This is supported because the pre-metamorphic stage is a larval development period that requires a tight regulation of gene expression for specific ontogenesis [38].

Comparative analysis of the different approaches to cluster and analyze the DEGs obtained (i.e., hard clustering, soft clustering and overlapping clustered DEGs by custom Venn diagram), revealed that both hierarchical and soft clustering highlight genes mostly involved in developmental processes during the pre-metamorphosis stage. At the climax stage, the three approaches revealed up-regulated genes associated to immune system functions. However, comparisons between hierarchical and soft clustering at the post-metamorphic stage showed different enriched ontologies. From hierarchical clustering, significant GO terms at post-metamorphic stage were related to immune system processes as found in the metamorphic climax. Soft clustering exhibited stage-specific GO terms and, thus, this approach led to an increased resolution to identify stage-specific gene expression and enriched ontologies across turbot metamorphosis. For this reason, we focused on the data analyzed by soft clustering approach.

At the onset of the metamorphic climax (30 dpf), a strong morphological remodelling occurs, including the beginning of the migration of the right eye. As expected, our results showed significant transcriptional activation of thyroid hormone receptor alpha-A (thraa) and thyroid-stimulating hormone beta subunit (tshba) genes at this specific stage. All studies to date suggest that THs play a key role in the induction of the teleost metamorphosis process. Thus, metamorphosis in teleosts is triggered by the hypothalamic-pituitary-thyroid (HPT) axis, which is constituted of brain neuropeptide thyrotropin-releasing hormone (TRH), brain neuropeptide corticotropin-releasing hormone (CRH), pituitary glycoprotein hormone (thyrotropin, TSH) and THs (thyroxime [T4] and triiodothyronine [T3]). In some teleosts, such as coho salmon, it has been suggested that CRH, rather than TRH, plays a key role as a stimulator of TSH secretion by the pituitary gland [21,43]. Our results corroborate the critical role of THs in the regulation of the flatfish metamorphosis process. Interestingly, during this stage, one of the most enriched GO categories was innate immune system. A significant up-regulation was detected for the chitinase family genes [44,45]. In addition, several genes of innate immune response were also up-regulated, including the dhx58, ifih1, irf3 and irf7genes. ifih1 gene encodes the melanoma differentiation-associated protein 5 (Mda5). This protein increases the phosphorylation levels of the transcription interferon regulatory factors 3 and 7 (irf3 and irf7), activating the expression of the type 1 interferon genes (ifnα and ifnβ) and initiating the processes of inflammation and cell death [46]. Mda5 helicase is activated by both endogenous and exogenous double-stranded RNA and several studies demonstrate that the regulation of Mda5 expression is linked to the induction of autoimmunity [47]. This suggests that Mda5 activation is not only due to the antiviral response but may also be stimulated by endogenous factors. It is well known that diverse innate immunity-related molecules are also expressed in the brain and play important roles in brain development [48]. We also observed an overexpression of casp10 and ripk3, which stimulate cell apoptosis and inflammation [49]. The results obtained by applying more restrictive statistical conditions also show a significant up-regulation of genes that enrich the immune response ontologies, such as mx, which promotes cell apoptosis [50], or faslg, which induces apoptosis in T cells [51]. Another enriched GO term at the onset of the metamorphic climax stage was regulation of T-cell migration. It is generally believed that the development of an immune response involves T-cell activation in lymphoid organs and subsequent migration to peripheral tissues to mediate tissue damage inflammation [52]. However, it has recently been shown that, in addition to the defense function of cells and immune molecules, they also play a key role in neurodevelopmental processes [48].

Our data show that during the climax stage of the developmental metamorphosis process in turbot brains, components of the immune system and THs could play an important role, as well as the processes of inflammation and cell death in the metamorphosis process at the brain level. Other studies in Xenopus also highlight the reorganization of the immune system during metamorphosis due to the need to replace larval tissue in adults and to support all the new chemical and biological products generated during this transformation [53,54].

At the post-metamorphic stage (57dpf), and after successfully completing the metamorphosis process, both morphological and behavioral changes were observed in the juvenile turbot. At this stage, one of the most enriched GO terms was dopamine biosynthetic process. A significant up-regulation was detected for nuclear receptor subfamily 4 (nr4a1) and GTP cyclohydrolase 1 (gch1) family genes. Dopamine is a neurotransmitter involved in the inhibitory control of TSH secretion [55]. The timeline profile of tshb expression during the metamorphosis process shows a significant increase during climax stage; however, significant high levels of tshb remain after the end of this stage. This may be because sufficiently high levels of dopamine have not yet been reached to inhibit tshb expression. Another enriched GO term induced at the post-metamorphic stage was regulation of removal of superoxide radicals. It is well known that the active processes of inflammation and apoptosis found at the climax stage generate free radicals that must be removed to avoid further damage in the organism at later stages [56]. 041b061a72


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