Melanopsin elevates locomotor activity during the wake state of the diurnal zebrafish
2022; Springer Nature; Volume: 23; Issue: 5 Linguagem: Inglês
10.15252/embr.202051528
ISSN1469-3178
AutoresMarcus P. S. Dekens, Bruno M. Fontinha, Miguel Gallach, Sandra Pflügler, Kristin Tessmar‐Raible,
Tópico(s)Sleep and Wakefulness Research
ResumoArticle1 March 2022Open Access Source DataTransparent process Melanopsin elevates locomotor activity during the wake state of the diurnal zebrafish Marcus P S Dekens Corresponding Author Marcus P S Dekens [email protected] orcid.org/0000-0003-1689-3491 Max Perutz Laboratory, Centre for Molecular Biology, University of Vienna and Medical University of Vienna, Vienna, Austria Contribution: Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Bruno M Fontinha Bruno M Fontinha orcid.org/0000-0003-1670-2667 Max Perutz Laboratory, Centre for Molecular Biology, University of Vienna and Medical University of Vienna, Vienna, Austria Contribution: Investigation Search for more papers by this author Miguel Gallach Miguel Gallach orcid.org/0000-0001-7002-310X Max Perutz Laboratory, Centre for Molecular Biology, University of Vienna and Medical University of Vienna, Vienna, Austria Max Perutz Laboratory, Centre for Integrative Bioinformatics, University of Vienna and Medical University of Vienna, Vienna, Austria Contribution: Formal analysis Search for more papers by this author Sandra Pflügler Sandra Pflügler orcid.org/0000-0002-1158-5169 Max Perutz Laboratory, Centre for Molecular Biology, University of Vienna and Medical University of Vienna, Vienna, Austria Contribution: Investigation Search for more papers by this author Kristin Tessmar-Raible Kristin Tessmar-Raible orcid.org/0000-0002-8038-1741 Max Perutz Laboratory, Centre for Molecular Biology, University of Vienna and Medical University of Vienna, Vienna, Austria Research Platform "Marine Rhythms of Life", University of Vienna, Vienna, Austria Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Project administration, Writing - review & editing Search for more papers by this author Marcus P S Dekens Corresponding Author Marcus P S Dekens [email protected] orcid.org/0000-0003-1689-3491 Max Perutz Laboratory, Centre for Molecular Biology, University of Vienna and Medical University of Vienna, Vienna, Austria Contribution: Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Bruno M Fontinha Bruno M Fontinha orcid.org/0000-0003-1670-2667 Max Perutz Laboratory, Centre for Molecular Biology, University of Vienna and Medical University of Vienna, Vienna, Austria Contribution: Investigation Search for more papers by this author Miguel Gallach Miguel Gallach orcid.org/0000-0001-7002-310X Max Perutz Laboratory, Centre for Molecular Biology, University of Vienna and Medical University of Vienna, Vienna, Austria Max Perutz Laboratory, Centre for Integrative Bioinformatics, University of Vienna and Medical University of Vienna, Vienna, Austria Contribution: Formal analysis Search for more papers by this author Sandra Pflügler Sandra Pflügler orcid.org/0000-0002-1158-5169 Max Perutz Laboratory, Centre for Molecular Biology, University of Vienna and Medical University of Vienna, Vienna, Austria Contribution: Investigation Search for more papers by this author Kristin Tessmar-Raible Kristin Tessmar-Raible orcid.org/0000-0002-8038-1741 Max Perutz Laboratory, Centre for Molecular Biology, University of Vienna and Medical University of Vienna, Vienna, Austria Research Platform "Marine Rhythms of Life", University of Vienna, Vienna, Austria Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Project administration, Writing - review & editing Search for more papers by this author Author Information Marcus P S Dekens *,1, Bruno M Fontinha1, Miguel Gallach1,2,4, Sandra Pflügler1 and Kristin Tessmar-Raible1,3 1Max Perutz Laboratory, Centre for Molecular Biology, University of Vienna and Medical University of Vienna, Vienna, Austria 2Max Perutz Laboratory, Centre for Integrative Bioinformatics, University of Vienna and Medical University of Vienna, Vienna, Austria 3Research Platform "Marine Rhythms of Life", University of Vienna, Vienna, Austria 4Present address: iLabSystems, Vienna, Austria *Corresponding author. E-mail: [email protected] EMBO Reports (2022)23:e51528https://doi.org/10.15252/embr.202051528 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mammalian and fish pineals play a key role in adapting behaviour to the ambient light conditions through the release of melatonin. In mice, light inhibits nocturnal locomotor activity via the non-visual photoreceptor Melanopsin. In contrast to the extensively studied function of Melanopsin in the indirect regulation of the rodent pineal, its role in the intrinsically photosensitive zebrafish pineal has not been elucidated. Therefore, it is not evident if the light signalling mechanism is conserved between distant vertebrates, and how Melanopsin could affect diurnal behaviour. A double knockout of melanopsins (opn4.1-opn4xb) was generated in the diurnal zebrafish, which manifests attenuated locomotor activity during the wake state. Transcriptome sequencing gave insight into pathways downstream of Melanopsin, implying that sustained repression of the melatonin pathway is required to elevate locomotor activity during the diurnal wake state. Moreover, we show that light induces locomotor activity during the diurnal wake state in an intensity-dependent manner. These observations suggest a common Melanopsin-driven mechanism between zebrafish and mammals, while the diurnal and nocturnal chronotypes are inversely regulated downstream of melatonin. Synopsis This study proposes a correlation between the number of detected photons and locomotor activity in the diurnal wake state in zebrafish. Light elevates the activity level through a Melanopsin-mediated repression of the melatonin pathway. Melanopsin elevates locomotor activity during the diurnal wake state. Sustained repression of the melatonin pathway is required to maintain a high level of locomotor activity during the diurnal wake state. A common Opn4/OPN4-driven mechanism exists in zebrafish and mammals. Melanopsin regulates the immediate behavioural response to loss of illumination in zebrafish. The light level affects locomotor activity during the wake state. Introduction To adapt to the ambient light conditions, activity is regulated directly by light and indirectly through the circadian clock (Dunlap et al, 2004), which anticipates daily recurring events. In nocturnal mice, the non-visual photoreceptor Melanopsin (OPN4) functions in circadian clock entrainment (Panda et al, 2002; Ruby et al, 2002) and in the direct induction of sleep by light (Mrosovsky & Hattar, 2003; Lupi et al, 2008; Tsai et al, 2009). In contrast to nocturnal mice, where Opn4 is solely expressed in the intrinsically photoreceptive retinal ganglion cells (ipRGCs) (Hatori & Panda, 2010), several diurnal species also express Opn4 in the brain. Opn4 is transcribed in many domains of the human brain (Hawrylycz et al, 2012; Nissilä et al, 2017), the zebrafish larval brain (Davies et al, 2011; Matos-Cruz et al, 2011), the chicken pineal (Holthues et al, 2004; Bailey & Cassone, 2005; Chaurasia et al, 2005) and in the pineal of the Atlantic halibut (Eilertsen et al, 2014). In vertebrates, the pineal gland or epiphysis cerebri plays a key role in synchronising behaviour and physiology to the environmental light–dark (LD) cycles through the rhythmic production of the indolamine melatonin (Sapède & Cau, 2013). The melatonin level reaches its peak during the night in both diurnal and nocturnal animals (Challet, 2007), thus melatonin is a dark phase indicator. In mice, ipRGCs provide the central clock located in the suprachiasmatic nucleus (SCN) with photic information, which in succession regulates the pineal via the paraventricular nucleus (PVN) (Simonneaux & Ribelayga, 2003). In contrast to the mammalian pineal, which receives indirect photic input through sympathetic norepinephrine (NE) innervation (Simonneaux & Ribelayga, 2003), the pineals of many fish, amphibians, reptiles and birds display all characteristics of a photoreceptive organ (Korf et al, 1998; Ziv et al, 2007; Sapède & Cau, 2013; Ben-Moshe Livne et al, 2016; Bertolesi & McFarlane, 2018) and generate endogenous endocrine rhythms, combining input and output functions in the photoreceptor cell (Falcon et al, 2007). Moreover, NE does not play a role in regulating melatonin synthesis in zebrafish (Cahill, 1997). Melatonin is synthesised by four enzymes: Tryptophan hydroxylase (TPH) catalyses the oxidation of the amino acid tryptophan to 5-hydroxy-L-tryptophan followed by the removal of a carboxyl group by Dopa decarboxylase (DDC) to produce serotonin. Arylalkylamine N-acetyltransferase (AANAT) adds an acetyl group to produce N-acetyl-serotonin, which is then methylated by Acetyl-serotonin O-methyltransferase (ASMT) into melatonin. The nightly release of NE in the rodent pineal activates the cAMP pathway resulting in the phosphorylation of cAMP response element-binding protein (CREB), which induces Aanat transcription (Rohde et al, 2014a). Homeodomain transcription factors control pinealocyte specific gene expression by binding to highly conserved pineal regulatory elements (PIRE) (Li et al, 1998), thereby permitting pCREB to regulate these genes through CRE elements. The transcription factor Cone-rod homeodomain (CRX) induces Aanat (Li et al, 1998; Rohde et al, 2014b), Tph1 and Asmt (Rohde et al, 2019) transcription in the mature rat pineal. Furthermore, Orthodenticle homeobox 2 (OTX2) transactivates Crx (Nishida et al, 2003; Rohde et al, 2019) and induces Tph1, Aanat and Asmt (Rohde et al, 2019), and LIM homeobox 4 (LHX4) induces Aanat (Hertz et al, 2020). In zebrafish, the homeodomain transcription factor Otx5 has been reported to regulate aanat2 (Gamse et al, 2002). As the aanat2 promoter contains CRE elements (Falcon et al, 2007), Creb could also play a role in regulating the zebrafish melatonin pathway. The rate of melatonin synthesis mainly depends on AANAT (Klein & Weller, 1970; Klein et al, 1997). However, the melatonin level is effectively raised by inducing multiple components of the melatonin pathway (Liu & Borjigin, 2005; Rohde et al, 2019). Melatonin has been demonstrated to promote sleep in diurnal vertebrates ranging from zebrafish (Zhdanova et al, 2001) to humans (Dollins et al, 1994; Mintz et al, 1998; Zhdanova et al, 2002; Brzezinski et al, 2005; Lok et al, 2019), and a light pulse in the night suppresses melatonin production with the strongest effect at the wavelength where Opn4/OPN4 has its absorption optimum in both zebrafish (Ziv et al, 2007) and humans (Lewy et al, 1980; Czeisler et al, 1995; Cajochen et al, 2000; Lockley et al, 2003). The reduction in the melatonin level is proportional to the intensity of the light pulse (Max & Menaker, 1992; Zachmann et al, 1992b; Bolliet et al, 1995) implying regulation by a light intensity detector. Therefore, OPN4 is a primary candidate in this process (Wong et al, 2005; Mure et al, 2016). Several human studies have shown that bright light during daytime increases activity and vigilance (Cajochen et al, 2000; Phipps-Nelson et al, 2003; Lockley et al, 2006; Smolders et al, 2012; Smolders & de Kort, 2014; Knaier et al, 2016). These data suggest that in diurnal vertebrates, suppression of melatonin production by light may also play a role in regulating activity levels during the wake state. To investigate how zebrafish adapt to ambient light conditions and the role of Opn4 in this process, a double knockout (dko) was generated of opn4.1 and opn4xb which are coexpressed in the pineal. This study implies that locomotor activity during the wake state is regulated by an Melanopsin-driven mechanism that is common between mammals and zebrafish despite the differences in light input and chronotypes. The Opn4-dependent transcriptome also suggests that Opn4 influences the immune system and the cell cycle in addition to the control it exerts over genes that encode melatonin synthesis enzymes. These findings alter the perspective of a photoreceptor that has until now solely been associated with behaviour to one that adapts diverse functions to the environment. Results opn4 expression in the brain and knockout strategy The expression patterns of all five opn4 homologs were characterised in the adult zebrafish brain by in situ hybridisation (ish). The opn4 genes show broad expression in the adult brain (Fig EV1A–E, Table EV1), which we documented in accordance with the neuroanatomical terminology established by Wullimann (Wullimann et al, 1996; Baeuml et al, 2019). opn4.1 and opn4xb stand out as the opn4 genes that are strongly expressed in the pineal (Fig 1A and B). To knockout opn4.1 and opn4xb the transcription activator-like effector nuclease (TALEN) genome editing technique (Cermak et al, 2011; Bedell et al, 2012) was applied. A premature stop codon was introduced close to the start codon in each gene (Fig 1C and D, Table 1). Opn4 is a seven transmembrane G-protein coupled receptor with a light absorbing moiety, the chromophore retinal, bound to helix seven (Fig 1E). The mutated Opn4.1 has lost all transmembrane helices, and only the first and second transmembrane helices remain in the mutated Opn4xb. Thus, from the structure–function relationship, it can be deduced that neither of the mutated opn4 genes encodes a functional photoreceptor. A homozygous double knockout (dko) was generated, as both Melanopsins are likely to have redundant functions given their conspicuous coexpression. Click here to expand this figure. Figure EV1. ishs on brain sections reveal broad expression of all five zebrafish melanopsins in the mature brain (A–E) opn4.1 (A) opn4a (B) opn4b (C) opn4xa (D) and opn4xb (E). A horizontal line through a representation of the brain, above the section, indicates the location of the section within the brain. The names and abbreviations of all brain domains in which opn4 is expressed are listed in Table EV1. Note that the duration of the colorimetric development of the ishs presented in this figure is not exactly the same. Scale bar: 1.0 mm. Download figure Download PowerPoint Figure 1. melanopsin expression in the brain and knockout strategy A, B. In situs on adult brain sections reveal that (A) opn4.1 and (B) opn4xb are coexpressed in many brain domains including the epiphysis cerebri (indicated with E) or pineal. A horizontal line through a representation of the brain, above the section, indicates the location of the section within the brain. Scale bar: 1.0 mm. The brain domains in which the five melanopsins are expressed are presented in Fig EV1 and Table EV1. C. The TALEN genome-editing technique was applied for site-directed mutagenesis. A 5 bp deletion was introduced proximal to the start codon of opn4.1, resulting in a frame shift and stop codon. The grey box indicates the coding sequence, the red line indicates the location of the premature stop codon and the grey triangle dispays the wild-type sequence aligned with the mutated sequence. D. An 8 bp deletion was introduced in the second exon (grey box) of the opn4xb gene resulting in a premature stop codon. E. Model of the seven transmembrane G-protein-coupled photoreceptor shows the light absorbing molecule, the chromophore retinal (yellow), bound to helix 7 (blue). The red crosses indicate where the Melanopsins in the mutants are truncated: the mutated Opn4.1 has lost all transmembrane helices and the mutated Opn4xb ends after the second transmembrane helix. From the structure–function relationship of the photoreceptor, one can deduce that neither of the mutant Melanopsins is functional. Download figure Download PowerPoint Table 1. Transcription activator-like effector DNA-binding domains. Ensembl ID Gene Target Site TAL1 DNA Binding Domain TAL2 DNA Binding Domain ENSDARG00000007553 opn4.1 proximal to START codon tcactgtgcccctggagacat ttcccaaagattccttaaag ENSDARG00000103259 opn4xb exon 2 tctccaatcttcttcatca ttctccaaacatccact Expression profiling reveals pathways downstream of Opn4 Transcriptome sequencing was applied on cDNA from eyes and brain parts of wild-type and opn4 dko to reveal genes that act downstream of Opn4. For this purpose mature fish were used, as it is not possible to dissect larval brains due to their small size. The fish were entrained for 14 days to a 12:12 h LD regime, followed by dissection of the eyes and brains in the light phase at ZT4-6 (Zeitgeber Time). The brains were separated in an anterior and posterior part (Fig 2A) to reduce complexity. The anterior brain consists of a part of the forebrain including the pineal. The posterior brain consists of the mid- and hindbrain and the remaining part of the forebrain, which comprises the whole hypothalamus and pretectum, and part of the epithalamus (i.e. habenula), thalamus and posterior tuberculum. The olfactory bulbs and pituitary were omitted. Following transcriptome sequencing the read counts were analysed with edgeR software in which the cutoff for differential expression was set at a significance level of α = 0.05 and the false discovery rate (FDR) was applied (Benjamini & Hochberg, 1995). Most differentially expressed genes are in the brain: 284 in the anterior brain, 139 in the posterior brain and 16 in the eye (Fig 2B, Appendix Fig S1A–C). Next, the differentially expressed genes were analysed with KEGG software to identify clusters of gene products that act in the same pathway. This associated the differentially expressed genes from the opn4 dko anterior brain with several pathways (Source Data). The largest clusters of genes were assigned to phototransduction (dre04744: 14 genes) and metabolic (dre01100: 15 genes) pathways (Fig 2D, Appendix Fig S1A). Of the latter, 5 genes were assigned to the subcategory tryptophan metabolism (dre00380): tph1a (P = 7.89 × 10−7), tph2 (P = 1.89 × 10−13), ddc (P = 4.05 × 10−11), aanat2 (P = 0.04) and asmt (P = 4.74 × 10−38). These encode all the enzymes required for melatonin synthesis (Fig 3A and C, Appendix Fig S1D–F). Many of the differentially expressed genes that were assigned to the phototransduction pathway have been reported to be expressed in the zebrafish pineal: saga (P = 2.46 × 10−97), gngt1 (P = 1.56 × 10−32), pde6gb (P = 3.71 × 10−11) [Thisse et al, ZFIN direct data submission], grk7a (P = 6.32 × 10−11) (Rinner et al, 2005), grk7b (P = 0.047) (Rinner et al, 2005), rcvrna (P = 2.32 × 10−11) (Zang et al, 2015), rcvrn2 (P = 1.21 × 10−5) (Zang et al, 2015) and gnat1 (P = 8.52 × 10−22) (Lagman et al, 2015). In the posterior brain, two differentially expressed genes are linked to phototransduction: gnat1 (P = 0.005) and saga (P = 0.004), and one to tryptophan metabolism: asmt (P = 0.001) (Appendix Fig S1B), which are also overexpressed in the anterior brain. Because the stalk of the pineal extends into the posterior brain, a small part of its specific transcriptome could be included in this data set. Fisher's exact test (Fisher, 1922) indicates that differential expression of the same genes in the anterior and posterior brain is unlikely due to coincidence (Fig 2C). The large number of common differentially expressed genes may well be the result of shared forebrain regions between these data sets. All data sets were also analysed for functional association with FuncAssociate software, applying a cutoff at the significance level α = 0.05. This revealed gene ontology (GO) attributes for differentially expressed genes of the eye and anterior brain (Source Data). The GO attributes of the latter are all related to the effect of light on biological processes (Fig 2E). Most of the genes that were associated with light-dependent processes are expressed in the zebrafish pineal. These are rbp4l (P = 3.85 × 10−60), arr3a (P = 1.02 × 10−26), rpe65a (P = 9.44 × 10−15), crx (P = 2.69 × 10−13) (Gamse et al, 2002), stra6 (P = 1.14 × 10−5), rlbp1a (P = 0.001), aanat2, pde6gb [Thisse et al, ZFIN direct data submission], grk7a (Rinner et al, 2005), grk7b (Rinner et al, 2005), rcvrna (Zang et al, 2015), rcvrn2 (Zang et al, 2015), rcvrn3 (P = 1.28 × 10−45) (Zang et al, 2015), gnat1 (Lagman et al, 2015), rbp3 (P = 6.87 × 10−15) (Nickerson et al, 2006), irbpl (P = 0.012) (Nickerson et al, 2006), unc119.2 (P = 0.0003) (Toyama et al, 2009) and pp2 (P = 1.23 × 10−6) (Koyanagi et al, 2015). Also otx5 (P = 0.001) and lhx4 (P = 0.003) (Appendix Fig S1A,G,I) have been demonstrated to be expressed in the pineal (Gamse et al, 2002; Weger et al, 2016). Note that all the genes that are associated with the pineal are overexpressed in the opn4 dko anterior brain (Appendix Fig S1A). As the pineal produces melatonin and detects light (Klein, 2004; Sapède & Cau, 2013), the differentially expressed genes assigned to the melatonin and phototransduction pathways in the opn4 dko anterior brain (Fig 2D and E, Appendix Fig S1A) point to a defect in this gland. In addition to these pathways, five differentially expressed genes in the opn4 dko anterior brain were assigned to the mitogen-activated protein kinase (MAPK) pathway: ngfra (P = 0.042), dusp7 (P = 0.002), ppm1na (P = 1.92 × 10−6), daxx (P = 0.006), jund (P = 0.048) and several genes were assigned to a range of pathways with diverse biological functions (Source Data). Two differentially expressed genes in the eye: mhc1uba (P = 7.06 × 10−96) and mhc1uka (P = 3.05 × 10−7) (Appendix Fig S1C,P and Q) were both associated with the GO attributes of antigen processing and presentation of peptide antigen (GO:0048002) and antigen binding (GO:0003823). Importantly, a substantial number of differentially expressed genes in the opn4 dko function in the immune response: mhc1uba (Pa = 3.46 × 10−185, Pp = 7.89 × 10−210), mhc1uka (Pa = 0.04), caspb (Pa = 0.0005, Pp = 4.89 × 10−9), caspbl (Pa = 0.046), ly6pge (Pa = 3.10 × 10−9), nrp1b (Pa = 0.027), mpeg1.1 (Pa = 0.049), mrc1 (Pa = 0.012), pigrl3.5 (Pa = 0.042), dicp3.3 (Pp = 0.0008), tap2a (Pp = 0.037), timd4 (Pp = 0.002), cd74b (Pp = 0.015), b2m (Pp = 0.047) and cell division: mcm7 (Pa = 2.15 × 10−13, Pp = 2.30 × 10−31) cdk9 (Pa = 3.45 × 10−11, Pp = 2.47 × 10−5), ccna2 (Pp = 0.040), cdk20 (Pp = 0.038) (Appendix Fig S1A–C, K–Q), implying that Opn4 controls diverse processes and confirming previous reports of cell cycle regulation by light (Dekens et al, 2003; Kowalska et al, 2013), regulation of the immune/inflammation response by the rat pineal (Bailey et al, 2009) and the role of melatonin in buffering the immune system (Carrillo-Vico et al, 2013). Figure 2. Expression profiling reveals pathways downstream of Opn4 Representation of the zebrafish adult brain shows the lateral and dorsal view with abbreviations for the following domains: olfactory bulb [OB], telencephalon [Tel], epiphysis cerebri or pineal [E], optic tectum [TeO], cerebellum [Ce], medulla oblongata [MO], hypothalamus [H] and hypophysis or pituitary [Pit]. Transcriptome sequencing was performed on cDNA from wild-type and opn4 dko eyes and brains, sampled in the light phase (ZT4-6). The brains were separated in an anterior part, which includes part of the forebrain and the pineal, and a posterior part, which includes part of the forebrain and the whole mid- and hindbrain. The cut lines through the depicted brain indicate where the brain was partitioned and which parts were subjected to transcriptome sequencing. The blue (anterior brain) and grey (posterior brain) arrows connect the brain parts in (A) with the differentially expressed genes in (B). Venn diagram shows the number of differentially expressed genes in the opn4 dko anterior brain (dark blue circle), posterior brain (grey circle) and eye (light blue circle). The cutoff for differential expression was set at the significance level of α = 0.05. Heat maps of all the differentially expressed genes are presented in Appendix Fig S1A–C. The number of differentially expressed genes that are shared between the data sets is indicated where the datasets overlap. Fisher's exact test shows that the shared differentially expressed genes between data sets are unlikely to be the result of coincidence. P-values for the common differentially expressed genes are indicated where the datasets overlap. The large number of common differentially expressed genes between the data sets derived from the anterior and posterior brain parts is likely due to forebrain regions that are shared between these parts. Pie chart shows the number of differentially expressed genes in the opn4 dko anterior brain that were assigned with KEGG software to phototransduction (dark blue), metabolic (orange) and other pathways. Of the metabolic pathway, 5 genes were assigned to the subcategory tryptophan metabolism (red), which encode all the enzymes that convert tryptophan into melatonin. Note that 189 genes were not assigned to a pathway. Ancestor charts show the gene ontology attributes to which the differentially expressed genes in the opn4 dko anterior brain were assigned with FuncAssociate software (cutoff: α = 0.05). The number of genes assigned to an attribute is indicated in the upper right corner. All retrieved biological processes and molecular functions are associated with light, consistent with knockout of a photoreceptor. Source data are available online for this figure. Source Data for Figure 2 [embr202051528-sup-0007-SDataFig2.xlsx] Download figure Download PowerPoint Figure 3. Melatonin pathway genes are overexpressed in the opn4 dko anterior brain Transcriptome sequencing reveals a significant higher number of ddc reads in the opn4 dko than in the wild-type anterior brain, and no difference in the posterior brain and eyes. ddc reads were normalised to 1,000 actb1 reads. qPCR confirms a significant higher ddc transcript level in the opn4 dko (grey box) than in the wild-type (blue box) anterior brain. In contrast to the transcriptome data, a significant higher level of ddc was detected by qPCR in the opn4 dko eyes. Quantification of the ddc mRNA level is relative to the actb1 mRNA measured in the sample. Chart shows normalised read counts for asmt. A significant higher number of asmt reads is detected in the opn4 dko than in the wild-type anterior brain. Note that asmt mRNA is detected in the posterior brain because the pineal stalk is most likely included in this part. qPCR confirms a significant higher asmt transcript level in the opn4 dko than in the wild-type anterior brain. Quantification of the asmt mRNA level is relative to the actb1 mRNA measured in the sample. Data information: In (A) and (C), blue markers indicate wild-type, grey markers indicate opn4 dko and red bar indicates mean. Biological replicates are indicated with round, triangular and square markers (n = 3). Asterisks indicate significance (0.01 < P(*) < 0.05, 0.001 < P(**) < 0.01, P(***) < 0.001, ns = not significant). In (B) and (D), boxplot divides the data in quartiles: the box indicates the interquartile range, with the horizontal line in the box denoting the median of the data set, the whiskers extend to the minimum and maximum, and meet the box at the median of the lower (quartile 1) and median of the upper (quartile 3) half of the dataset. Black dots indicate biological replicates (n = 12), the red dot indicates the mean and red error bars indicate the confidence interval (95%). Download figure Download PowerPoint asmt is overexpressed in the opn4 dko pineal To validate the transcriptome sequencing data, we repeated the same entrainment and sampling procedure and determined the levels of ddc and asmt mRNA by qPCR, which confirmed significant overexpression of ddc (Fig 3B U-test: P = 4.42 × 10−5) and asmt (Fig 3D U-test: P = 0.0004) in the mature opn4 dko anterior brain. Both ddc and asmt transcripts are expressed in the mature wild-type and opn4 dko pineals, as demonstrated by ish (Fig EV2A–D). The elevated asmt mRNA levels detected in the opn4 dko brain (Fig 3C and D) can be attributed to the pineal, as asmt is solely expressed in this gland and not ectopically expressed in the opn4 dko brain. Note that asmt is not overexpressed in the eye (Fig 3C and D). Click here to expand this figure. Figure EV2. ish with ddc or asmt probe on opn4 dko mature brains show wild-type expression patterns A, B. Same ddc expression pattern was observed in wild-type (A) and opn4 dko (B) brains (pineal lost from the brain slices in (B) centre panels). C, D. asmt expression in wild-type (C) and opn4 dko (D) adult brains show that asmt is not ectopically expressed. Thus, the higher asmt transcript level measured with qPCR in the opn4 dko anterior brain can be attributed to the pineal. Data information: The horizontal line through a representation of the brain, above the section, indicates the location of the section within the brain. Abbreviations: epiphysis cerebri (pineal) [E], caudal zone of periventricular hypothalamus [Hc], inferior raphe [IR], periventricular pretectum [Pr], preoptic area [PO], posterior tuberculum [PT], paraventricular organ [PVO], suprachiasmatic nucleus [SCN], superior raphe [SR]. Note that the duration of the colorimetric development of the ishs in this figure is not exactly the same. Scale bar: 1.0 mm. Downl
Referência(s)