Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Brain transcriptome profile after CRISPR-induced ghrelin mutations in zebrafish

Abstract

Ghrelin (GRL) is a gut-brain hormone with a role in a wide variety of physiological functions in mammals and fish, which points out the ghrelinergic system as a key element for the appropriate biological functioning of the organism. However, many aspects of the multifunctional nature of GRL remain to be better explored, especially in fish. In this study, we used the CRISPR/Cas9 genome editing technique to generate F0 zebrafish in which the expression of grl is compromised. Then, we employed high-throughput mRNA sequencing (RNA-seq) to explore changes in the brain transcriptome landscape associated with the silencing of grl. The CRISPR/Cas9 technique successfully edited the genome of F0 zebrafish resulting in individuals with considerably lower levels of GRL mRNAs and protein and ghrelin O-acyl transferase (goat) mRNAs in the brain, intestine, and liver compared to wild-type (WT) zebrafish. Analysis of brain transcriptome revealed a total of 1360 differentially expressed genes (DEGs) between the grl knockdown (KD) and WT zebrafish, with 664 up- and 696 downregulated DEGs in the KD group. Functional enrichment analysis revealed that DEGs are highly enriched for terms related to morphogenesis, metabolism (especially of lipids), entrainment of circadian clocks, oxygen transport, apoptosis, and response to stimulus. The present study offers valuable information on the central genes and pathways implicated in functions of GRL, and points out the possible involvement of this peptide in some novel functions in fish, such as apoptosis and oxygen transport.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Anders S, Pyl PT, Huber W (2015) HTSeq—a Python framework to work with high-throughput sequencing data. Bioinforma Oxf Engl 31:166–169. https://doi.org/10.1093/bioinformatics/btu638

  2. Asakawa A, Inui A, Kaga T, Katsuura G, Fujimiya M, Fujino MA, Kasuga M (2003) Antagonism of ghrelin receptor reduces food intake and body weight gain in mice. Gut 52:947–952

  3. Bali A, Jaggi AS (2016) An integrative review on role and mechanisms of ghrelin in stress, anxiety and depression. Curr Drug Targets 17:495–507

  4. Bewick GA, Kent A, Campbell D, Patterson M, Ghatei MA, Bloom SR, Gardiner JV (2009) Mice with hyperghrelinemia are hyperphagic and glucose intolerant and have reduced leptin sensitivity. Diabetes 58:840–846. https://doi.org/10.2337/db08-1428

  5. Blanco AM, Gómez-Boronat M, Alonso-Gómez ÁL, Yufa R, Unniappan S, Delgado MJ, Valenciano AI (2017) Characterization of ghrelin O-acyltransferase (GOAT) in goldfish (Carassius auratus). PLoS One 12:e0171874. https://doi.org/10.1371/journal.pone.0171874

  6. Bougarne N, Weyers B, Desmet SJ, Deckers J, Ray DW, Staels B, de Bosscher K (2018) Molecular actions of PPARα in lipid metabolism and inflammation. Endocr Rev 39:760–802. https://doi.org/10.1210/er.2018-00064

  7. Breves JP, Veillette PA, Specker JL (2009) Ghrelin in the summer flounder: immunolocalization to the gastric glands and action on plasma cortisol levels. Comp Biochem Physiol A Mol Integr Physiol 152:268–272. https://doi.org/10.1016/j.cbpa.2008.10.020

  8. Charpentier E, Doudna JA (2013) Biotechnology: rewriting a genome. Nature 495:50–51. https://doi.org/10.1038/495050a

  9. Cruz SA, Tseng Y-C, Kaiya H, Hwang PP (2010) Ghrelin affects carbohydrate-glycogen metabolism via insulin inhibition and glucagon stimulation in the zebrafish (Danio rerio) brain. Comp Biochem Physiol A Mol Integr Physiol 156:190–200. https://doi.org/10.1016/j.cbpa.2010.01.019

  10. Date Y, Kojima M, Hosoda H, Sawaguchi A, Mondal MS, Suganuma T, Matsukura S, Kangawa K, Nakazato M (2000) Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 141:4255–4261. https://doi.org/10.1210/endo.141.11.7757

  11. Davies JS, Kotokorpi P, Eccles SR, Barnes SK, Tokarczuk PF, Allen SK, Whitworth HS, Guschina IA, Evans BAJ, Mode A, Zigman JM, Wells T (2009) Ghrelin induces abdominal obesity via GHS-R-dependent lipid retention. Mol Endocrinol Baltim Md 23:914–924. https://doi.org/10.1210/me.2008-0432

  12. De Smet B, Depoortere I, Moechars D et al (2006) Energy homeostasis and gastric emptying in ghrelin knockout mice. J Pharmacol Exp Ther 316:431–439. https://doi.org/10.1124/jpet.105.091504

  13. Dezaki K (2013) Ghrelin function in insulin release and glucose metabolism. In: Benso A, Casanueva FF, Ghigo E, Granata R (eds) Endocrine Development. S. KARGER AG, Basel, pp 135–143

  14. Dezaki K, Sone H, Koizumi M, Nakata M, Kakei M, Nagai H, Hosoda H, Kangawa K, Yada T (2006) Blockade of pancreatic islet-derived ghrelin enhances insulin secretion to prevent high-fat diet-induced glucose intolerance. Diabetes 55:3486–3493. https://doi.org/10.2337/db06-0878

  15. Diotel N, Servili A, Gueguen M-M, Mironov S, Pellegrini E, Vaillant C, Zhu Y, Kah O, Anglade I (2011) Nuclear progesterone receptors are up-regulated by estrogens in neurons and radial glial progenitors in the brain of zebrafish. PLoS One 6:e28375. https://doi.org/10.1371/journal.pone.0028375

  16. Duffield GE, Watson NP, Mantani A, Peirson SN, Robles-Murguia M, Loros JJ, Israel MA, Dunlap JC (2009) A role for Id2 in regulating photic entrainment of the mammalian circadian system. Curr Biol 19:297–304. https://doi.org/10.1016/j.cub.2008.12.052

  17. Ferrini F, Salio C, Lossi L, Merighi A (2009) Ghrelin in central neurons. Curr Neuropharmacol 7:37–49. https://doi.org/10.2174/157015909787602779

  18. Gagnon JA, Valen E, Thyme SB, Huang P, Ahkmetova L, Pauli A, Montague TG, Zimmerman S, Richter C, Schier AF (2014) Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PLoS One 9:e98186. https://doi.org/10.1371/journal.pone.0098186

  19. Gao S, Casals N, Keung W, Moran TH, Lopaschuk GD (2013) Differential effects of central ghrelin on fatty acid metabolism in hypothalamic ventral medial and arcuate nuclei. Physiol Behav 118:165–170. https://doi.org/10.1016/j.physbeh.2013.03.030

  20. Gnanapavan S, Kola B, Bustin SA, Morris DG, McGee P, Fairclough P, Bhattacharya S, Carpenter R, Grossman AB, Korbonits M (2002) The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. J Clin Endocrinol Metab 87:2988. https://doi.org/10.1210/jcem.87.6.8739

  21. Gutierrez JA, Solenberg PJ, Perkins DR, Willency JA, Knierman MD, Jin Z, Witcher DR, Luo S, Onyia JE, Hale JE (2008) Ghrelin octanoylation mediated by an orphan lipid transferase. Proc Natl Acad Sci U S A 105:6320–6325. https://doi.org/10.1073/pnas.0800708105

  22. Hatef A, Yufa R, Unniappan S (2015) Ghrelin O-acyl transferase in zebrafish is an evolutionarily conserved peptide upregulated during calorie restriction. Zebrafish 12:327–338. https://doi.org/10.1089/zeb.2014.1062

  23. Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JRJ, Joung JK (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31:227–229. https://doi.org/10.1038/nbt.2501

  24. Janzen WJ, Duncan CA, Riley LG (2012) Cortisol treatment reduces ghrelin signaling and food intake in tilapia, Oreochromis mossambicus. Domest Anim Endocrinol 43:251–259. https://doi.org/10.1016/j.domaniend.2012.04.003

  25. Jao L-E, Wente SR, Chen W (2013) Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci 110:13904–13909. https://doi.org/10.1073/pnas.1308335110

  26. Kaiya H, Kojima M, Hosoda H, Riley LG, Hirano T, Grau EG, Kangawa K (2003) Amidated fish ghrelin: purification, cDNA cloning in the Japanese eel and its biological activity. J Endocrinol 176:415–423

  27. Kaiya H, Miyazato M, Kangawa K, Peter RE, Unniappan S (2008) Ghrelin: a multifunctional hormone in non-mammalian vertebrates. Comp Biochem Physiol A Mol Integr Physiol 149:109–128. https://doi.org/10.1016/j.cbpa.2007.12.004

  28. Kamegai J, Tamura H, Shimizu T, Ishii S, Tatsuguchi A, Sugihara H, Oikawa S, Kineman RD (2004) The role of pituitary ghrelin in growth hormone (GH) secretion: GH-releasing hormone-dependent regulation of pituitary ghrelin gene expression and peptide content. Endocrinology 145:3731–3738. https://doi.org/10.1210/en.2003-1424

  29. Kang KS, Yahashi S, Matsuda K (2011a) The effects of ghrelin on energy balance and psychomotor activity in a goldfish model: an overview. Int J Pept 2011:171034–171039. https://doi.org/10.1155/2011/171034

  30. Kang KS, Yahashi S, Matsuda K (2011b) Central and peripheral effects of ghrelin on energy balance, food intake and lipid metabolism in teleost fish. Peptides 32:2242–2247. https://doi.org/10.1016/j.peptides.2011.05.006

  31. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K (1999) Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402:656–660. https://doi.org/10.1038/45230

  32. Kwon I, Choe HK, Son GH, Kim K (2011) Mammalian molecular clocks. Exp Neurobiol 20:18–28. https://doi.org/10.5607/en.2011.20.1.18

  33. Lawrence C (2007) The husbandry of zebrafish (Danio rerio): a review. Aquaculture 269:1–20. https://doi.org/10.1016/j.aquaculture.2007.04.077

  34. Lilleness BM, Frishman WH (2016) Ghrelin and the cardiovascular system. Cardiol Rev 24:288–297. https://doi.org/10.1097/CRD.0000000000000113

  35. Litwack G (2018) Steroid hormones. In: Human Biochemistry. Elsevier, Amsterdam pp 467–506

  36. Liu J, Lin H, Cheng P, Hu X, Lu H (2009) Effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocytes. J Huazhong Univ Sci Technol Med Sci Hua Zhong Ke Ji Xue Xue Bao Yi Xue Ying Wen Ban Huazhong Keji Daxue Xuebao Yixue Yingdewen Ban 29:227–230. https://doi.org/10.1007/s11596-009-0218-x

  37. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262

  38. López M, Lage R, Saha AK, Pérez-Tilve D, Vázquez MJ, Varela L, Sangiao-Alvarellos S, Tovar S, Raghay K, Rodríguez-Cuenca S, Deoliveira RM, Castañeda T, Datta R, Dong JZ, Culler M, Sleeman MW, Álvarez CV, Gallego R, Lelliott CJ, Carling D, Tschöp MH, Diéguez C, Vidal-Puig A (2008) Hypothalamic fatty acid metabolism mediates the orexigenic action of ghrelin. Cell Metab 7:389–399. https://doi.org/10.1016/j.cmet.2008.03.006

  39. Lowe R, Shirley N, Bleackley M, Dolan S, Shafee T (2017) Transcriptomics technologies. PLoS Comput Biol 13:e1005457. https://doi.org/10.1371/journal.pcbi.1005457

  40. Maere S, Heymans K, Kuiper M (2005) BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinforma Oxf Engl 21:3448–3449. https://doi.org/10.1093/bioinformatics/bti551

  41. Mitsui S (2001) Antagonistic role of E4BP4 and PAR proteins in the circadian oscillatory mechanism. Genes Dev 15:995–1006. https://doi.org/10.1101/gad.873501

  42. Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E (2014) CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res 42:W401–W407. https://doi.org/10.1093/nar/gku410

  43. Mori K, Yoshimoto A, Takaya K, Hosoda K, Ariyasu H, Yahata K, Mukoyama M, Sugawara A, Hosoda H, Kojima M, Kangawa K, Nakao K (2000) Kidney produces a novel acylated peptide, ghrelin. FEBS Lett 486:213–216

  44. Muccioli G, Lorenzi T, Lorenzi M, Ghè C, Arnoletti E, Raso GM, Castellucci M, Gualillo O, Meli R (2011) Beyond the metabolic role of ghrelin: a new player in the regulation of reproductive function. Peptides 32:2514–2521. https://doi.org/10.1016/j.peptides.2011.10.020

  45. Nisembaum LG, de Pedro N, Delgado MJ, Isorna E (2014) Crosstalking between the “gut-brain” hormone ghrelin and the circadian system in the goldfish. Effects on clock gene expression and food anticipatory activity. Gen Comp Endocrinol 205:287–295. https://doi.org/10.1016/j.ygcen.2014.03.016

  46. Osmundson TW, Eyre CA, Hayden KM, Dhillon J, Garbelotto MM (2013) Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding, genotyping, and disease diagnostics from fungal and oomycete samples. Mol Ecol Resour 13:66–74. https://doi.org/10.1111/1755-0998.12031

  47. Ozsolak F, Milos PM (2011) RNA sequencing: advances, challenges and opportunities. Nat Rev Genet 12:87–98. https://doi.org/10.1038/nrg2934

  48. Park JM, Kakimoto T, Kuroki T, Shiraishi R, Fujise T, Iwakiri R, Fujimoto K (2008) Suppression of intestinal mucosal apoptosis by ghrelin in fasting rats. Exp Biol Med Maywood NJ 233:48–56. https://doi.org/10.3181/0706-RM-169

  49. Perez-Tilve D, Hofmann SM, Basford J, Nogueiras R, Pfluger PT, Patterson JT, Grant E, Wilson-Perez HE, Granholm NA, Arnold M, Trevaskis JL, Butler AA, Davidson WS, Woods SC, Benoit SC, Sleeman MW, DiMarchi RD, Hui DY, Tschöp MH (2010) Melanocortin signaling in the CNS directly regulates circulating cholesterol. Nat Neurosci 13:877–882. https://doi.org/10.1038/nn.2569

  50. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308. https://doi.org/10.1038/nprot.2013.143

  51. Reed JA, Benoit SC, Pfluger PT, Tschöp MH, D'Alessio DA, Seeley RJ (2008) Mice with chronically increased circulating ghrelin develop age-related glucose intolerance. Am J Physiol-Endocrinol Metab 294:E752–E760. https://doi.org/10.1152/ajpendo.00463.2007

  52. Riley LG, Hirano T, Grau EG (2002) Rat ghrelin stimulates growth hormone and prolactin release in the tilapia, Oreochromis mossambicus. Zool Sci 19:797–800. https://doi.org/10.2108/zsj.19.797

  53. Riley LG, Fox BK, Kaiya H, Hirano T, Grau EG (2005) Long-term treatment of ghrelin stimulates feeding, fat deposition, and alters the GH/IGF-I axis in the tilapia, Oreochromis mossambicus. Gen Comp Endocrinol 142:234–240. https://doi.org/10.1016/j.ygcen.2005.01.009

  54. Salmerón C, Johansson M, Asaad M, Angotzi AR, Rønnestad I, Stefansson SO, Jönsson E, Björnsson BT, Gutiérrez J, Navarro I, Capilla E (2015) Roles of leptin and ghrelin in adipogenesis and lipid metabolism of rainbow trout adipocytes in vitro. Comp Biochem Physiol A Mol Integr Physiol 188:40–48. https://doi.org/10.1016/j.cbpa.2015.06.017

  55. Sánchez-Bretaño A, Blanco AM, Alonso-Gómez ÁL, Delgado MJ, Kah O, Isorna E (2017) Ghrelin induces clock gene expression in the liver of goldfish in vitro via protein kinase C and protein kinase a pathways. J Exp Biol 220:1295–1306. https://doi.org/10.1242/jeb.144253

  56. Shepperd E, Unniappan S (2011) Ghrelin regulates reproductive physiology in fish. Front Endocrinol 2. https://doi.org/10.3389/conf.fendo.2011.04.00067

  57. Shepperd E, Peng C, Unniappan S (2012) Ghrelinergic system in fish ovaries and ghrelin inhibition of germinal vesicle breakdown in zebrafish oocytes. Gen Comp Endocrinol 176:426–431. https://doi.org/10.1016/j.ygcen.2012.01.014

  58. Spencer SJ, Emmerzaal TL, Kozicz T, Andrews ZB (2015) Ghrelin’s role in the hypothalamic-pituitary-adrenal axis stress response: implications for mood disorders. Biol Psychiatry 78:19–27. https://doi.org/10.1016/j.biopsych.2014.10.021

  59. Sun Y, Butte NF, Garcia JM, Smith RG (2008) Characterization of adult ghrelin and ghrelin receptor knockout mice under positive and negative energy balance. Endocrinology 149:843–850. https://doi.org/10.1210/en.2007-0271

  60. Supek F, Bošnjak M, Škunca N, Šmuc T (2011) REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS One 6:e21800. https://doi.org/10.1371/journal.pone.0021800

  61. Tena-Sempere M (2008) Ghrelin as a pleotrophic modulator of gonadal function and reproduction. Nat Clin Pract Endocrinol Metab 4:666–674. https://doi.org/10.1038/ncpendmet1003

  62. Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinforma Oxf Engl 25:1105–1111. https://doi.org/10.1093/bioinformatics/btp120

  63. Tschöp M, Smiley DL, Heiman ML (2000) Ghrelin induces adiposity in rodents. Nature 407:908–913. https://doi.org/10.1038/35038090

  64. Uchida A, Zigman JM, Perelló M (2013) Ghrelin and eating behavior: evidence and insights from genetically-modified mouse models. Front Neurosci 7:121. https://doi.org/10.3389/fnins.2013.00121

  65. Unniappan S, Peter RE (2004) In vitro and in vivo effects of ghrelin on luteinizing hormone and growth hormone release in goldfish. Am J Phys Regul Integr Comp Phys 286:R1093–R1101. https://doi.org/10.1152/ajpregu.00669.2003

  66. Unniappan S, Canosa LF, Peter RE (2004) Orexigenic actions of ghrelin in goldfish: feeding-induced changes in brain and gut mRNA expression and serum levels, and responses to central and peripheral injections. Neuroendocrinology 79:100–108. https://doi.org/10.1159/000076634

  67. Velasco C, Librán-Pérez M, Otero-Rodiño C, López-Patiño MA, Míguez JM, Soengas JL (2016a) Intracerebroventricular ghrelin treatment affects lipid metabolism in liver of rainbow trout (Oncorhynchus mykiss). Gen Comp Endocrinol 228:33–39. https://doi.org/10.1016/j.ygcen.2016.01.016

  68. Velasco C, Librán-Pérez M, Otero-Rodiño C, López-Patiño MA, Míguez JM, Cerdá-Reverter JM, Soengas JL (2016b) Ghrelin modulates hypothalamic fatty acid-sensing and control of food intake in rainbow trout. J Endocrinol 228:25–37. https://doi.org/10.1530/JOE-15-0391

  69. Velasco C, Librán-Pérez M, Otero-Rodiño C, López-Patiño MA, Míguez JM, Soengas JL (2016c) Ceramides are involved in the regulation of food intake in rainbow trout (Oncorhynchus mykiss). Am J Phys Regul Integr Comp Phys 311:R658–R668. https://doi.org/10.1152/ajpregu.00201.2016

  70. Viani I, Vottero A, Tassi F, Cremonini G, Sartori C, Bernasconi S, Ferrari B, Ghizzoni L (2008) Ghrelin inhibits steroid biosynthesis by cultured granulosa-lutein cells. J Clin Endocrinol Metab 93:1476–1481. https://doi.org/10.1210/jc.2007-2063

  71. Wierup N, Svensson H, Mulder H, Sundler F (2002) The ghrelin cell: a novel developmentally regulated islet cell in the human pancreas. Regul Pept 107:63–69

  72. Wortley KE, Anderson KD, Garcia K, Murray JD, Malinova L, Liu R, Moncrieffe M, Thabet K, Cox HJ, Yancopoulos GD, Wiegand SJ, Sleeman MW (2004) Genetic deletion of ghrelin does not decrease food intake but influences metabolic fuel preference. Proc Natl Acad Sci U S A 101:8227–8232. https://doi.org/10.1073/pnas.0402763101

  73. Wren AM, Small CJ, Abbott CR, Dhillo WS, Seal LJ, Cohen MA, Batterham RL, Taheri S, Stanley SA, Ghatei MA, Bloom SR (2001) Ghrelin causes hyperphagia and obesity in rats. Diabetes 50:2540–2547

  74. Yada T, Dezaki K, Sone H, Koizumi M, Damdindorj B, Nakata M, Kakei M (2008) Ghrelin regulates insulin release and glycemia: physiological role and therapeutic potential. Curr Diabetes Rev 4:18–23

  75. Yang J, Brown MS, Liang G, Grishin NV, Goldstein JL (2008a) Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell 132:387–396. https://doi.org/10.1016/j.cell.2008.01.017

  76. Yang J, Zhao T-J, Goldstein JL, Brown MS (2008b) Inhibition of ghrelin O-acyltransferase (GOAT) by octanoylated pentapeptides. Proc Natl Acad Sci 105:10750–10755. https://doi.org/10.1073/pnas.0805353105

  77. Yannielli PC, Molyneux PC, Harrington ME, Golombek DA (2007) Ghrelin effects on the circadian system of mice. J Neurosci 27:2890–2895. https://doi.org/10.1523/JNEUROSCI.3913-06.2007

  78. Yoon M (2009) The role of PPARalpha in lipid metabolism and obesity: focusing on the effects of estrogen on PPARalpha actions. Pharmacol Res 60:151–159. https://doi.org/10.1016/j.phrs.2009.02.004

  79. Zhang C, Li L, Zhao B, Jiao A, Li X, Sun N, Zhang J (2016) Ghrelin protects against dexamethasone-induced INS-1 cell apoptosis via ERK and p38MAPK signaling. Int J Endocrinol 2016:1–11. https://doi.org/10.1155/2016/4513051

  80. Zhu X, Xu Y, Yu S, Lu L, Ding M, Cheng J, Song G, Gao X, Yao L, Fan D, Meng S, Zhang X, Hu S, Tian Y (2014) An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system. Sci Rep 4:6420. https://doi.org/10.1038/srep06420

Download references

Acknowledgments

This study was supported by a grant from the Spanish Ministry of Economy and Competitiveness (MINECO; AGL2016-74857-C3-2-R and AGL2016-74857-C3-3-R) to MJD and JMCR. AMB was a predoctoral fellow funded by the Spanish Ministry of Education and Science when this study was carried out.

Author information

Correspondence to José Miguel Cerdá-Reverter or María Jesús Delgado.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

José Miguel Cerdá-Reverter and María Jesús Delgado are equal co-senior contributing authors.

Electronic supplementary material

Fig. A1
figure7

PAGE electrophoresis showing the genotyping of grl sgRNA/Cas9-injected and uninjected zebrafish embryos based on the characterization of the duplex formation during DNA denaturation and annealing. Only homoduplexes are observed in the uninjected (control, CTRL) embryos, while homoduplexes and heteroduplexes are detected in the grl sgRNA/Cas9-injected (INJ). MM, molecular marker (PNG 51 kb)

High Resolution Image (TIF 406 kb)

Table A1

Differentially expressed genes between grl KD and WT fish after RNA-seq analysis (PDF 1412 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Blanco, A.M., Cortés, R., Bertucci, J.I. et al. Brain transcriptome profile after CRISPR-induced ghrelin mutations in zebrafish. Fish Physiol Biochem 46, 1–21 (2020). https://doi.org/10.1007/s10695-019-00687-6

Download citation

Keywords

  • Ghrelinergic system
  • CRISPR/Cas9
  • Knockdown
  • RNA-seq
  • Transcriptomic
  • Fish