Brain Structure and Function

, Volume 224, Issue 2, pp 627–642 | Cite as

Knockdown of calcium-binding calb2a and calb2b genes indicates the key regulator of the early development of the zebrafish, Danio rerio

  • Rahul C. Bhoyar
  • Arun G. JadhaoEmail author
  • Ankit Sabharwal
  • Gyan Ranjan
  • Sridhar Sivasubbu
  • Claudia Pinelli
Original Article


The present study initiates our investigation regarding the role of calb2a and calb2b genes that are expressed in the central nervous system, including the multiple tissues during early embryonic development of zebrafish. In this study, we have adopted individual and combined morpholino-mediated inactivation approach to investigate the functions of calb2a and calb2b in early development of the zebrafish. We have found that calb2a and calb2b morpholino alone failed to generate an obvious phenotype; however, morphological inspection in early developmental stages of calb2a and calb2b combined knockdown morphants show abnormal neural plate folding in midbrain-hindbrain region. In addition to this, combinatorial loss of these mRNA leads to severe hydrocephalus, axial curvature defect, and yolk sac edema in later developmental stages. Also, the combined knockdown of calb2a and calb2b are found to be associated with an impaired touchdown and swimming performance in the zebrafish. Co-injection of the calb2a and calb2b morpholino oligonucleotide cocktail with human CALB2 mRNA leads to the rescue of the strong phenotype. This study provided the first comprehensive analyses of the zebrafish Calb2a and Calb2b proteins; we have found that Calb2a and Calb2b are highly conserved across vertebrate species and originated from the same ancestral gene long back in the evolution. Homology modeling and docking with the similar structure and Ca2+ binding sites for both proteins provide the evidence that both the proteins may have similar function and one can compensate for the loss of other. Collectively, these findings confirm the unique and essential functions of calb2a and calb2b genes in the early development of the zebrafish.


Calb2a Calb2b Calcium-binding proteins Midbrain-hindbrain boundary Zebrafish 



Calbindin 2a


Calbindin 2b


Calbindin 2


Calcium-binding proteins




Morpholino oligonucleotide


Non-injected control


Central nervous system


Hours post fertilization


Day post fertilization


Midbrain-hindbrain boundary
















Protein database


Basic Local Alignment Search Tool



This work was supported by the fund from the Department of Biotechnology (DBT), Government of India (Project no. BT/PR4688/AAQ/03/585/2012) to AGJ. A donation of Axio Imager A2 (Zeiss) microscope by Alexander von Humboldt Foundation, Germany to AGJ, used for the photomicrography, is sincerely acknowledged.

Author contributions

All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: RCB, AGJ, SSB. Acquisition of data: RCB, SSB, AS. Analysis and interpretation of data: RCB, AGJ, AS, GR. Drafting of the manuscript: RCB, AGJ, CP, A.S, GR.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

429_2018_1797_MOESM1_ESM.pdf (46 kb)
Supplementary material 1 Supplementary Table 1 Validation results of selected homology model for Calb2a and Calb2b (PDF 46 KB)

Supplementary material 2 Movie 1 Non-injected control (NIC) embryos of 5 dpf stage showing the response to the mechanical stimuli by swimming away from the direction of the applied stimuli (FLV 461 KB)

Supplementary material 3 Movie 2 The combined calb2a and calb2b knockdown phenotype at 5 days post fertilization (dpf) development stage fails to respond to the applied stimuli and showing corkscrew-like swimming path (FLV 368 KB)


  1. Airaksinen MS, Eilers J, Garaschuk O, Thoenen H, Konnerth A, Meyer M (1997) Ataxia and altered dendritic calcium signaling in mice carrying a targeted null mutation of the calbindin D28k gene. Proc Natl AcadSci USA 94:1488–1493CrossRefGoogle Scholar
  2. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402CrossRefGoogle Scholar
  3. Amores A et al (1998) Zebrafish hox clusters and vertebrate genome evolution. Science 282:1711–1714CrossRefGoogle Scholar
  4. Andressen C, Blümcke I, Celio M (1993) Calcium-binding proteins: selective markers of nerve cells. Cell Tissue Res 271:181–208CrossRefGoogle Scholar
  5. Arendt O, Schwaller B, Brown EB, Eilers J, Schmidt H (2013) Restricted diffusion of calretinin in cerebellar granule cell dendrites implies Ca2+-dependent interactions via its EF-hand 5 domain. J Physiol 591:3887–3899CrossRefGoogle Scholar
  6. Baimbridge K, Miller J (1982) Immunohistochemical localization of calcium-binding protein in the cerebellum, hippocampal formation and olfactory bulb of the rat. Brain Res 245:223–229CrossRefGoogle Scholar
  7. Baimbridge K, Celio M, Rogers J (1992) Calcium-binding proteins in the nervous system. Trends Neurosci 15:303–308CrossRefGoogle Scholar
  8. Bang P, Yelick P, Malicki J, Sewell W (2002) High-throughput behavioral screening method for detecting auditory response defects in zebrafish. J Neurosci Methods 118:177–187CrossRefGoogle Scholar
  9. Benkert P, Tosatto SC, Schomburg D (2008) QMEAN: a comprehensive scoring function for model quality assessment. Proteins 71:261–277CrossRefGoogle Scholar
  10. Berdal A et al (1991) Differential expression of calbindin-D 28 kDa in rat incisor ameloblasts throughout enamel development. Anat Rec 230:149–163CrossRefGoogle Scholar
  11. Berman H, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) The protein data bank. Nucleic Acids Res 28:235–242CrossRefGoogle Scholar
  12. Bhoyar RC, Jadhao AG, Sivasubbu S, Singh AR, Sabharwal A, Palande NV, Biswas S (2017) Neuroanatomical demonstration of calbindin 2a- and calbindin 2b-like calcium binding proteins in the early embryonic development of zebrafish: mRNA study. Int J Dev Neurosci 60:26–33CrossRefGoogle Scholar
  13. Brand M et al (1996) Mutations affecting development of the midline and general body shape during zebrafish embryogenesis. Development 123:129–142Google Scholar
  14. Brockerhoff SE, Hurley JB, Janssen-Bienhold U, Neuhauss SC, Driever W, Dowling JE (1995) A behavioural screen for isolating zebrafish mutants with visual system defects. Proc Natl Acad Sci USA 92:10545–10549CrossRefGoogle Scholar
  15. Bronner F (1990) Intestinal calcium transport: the cellular pathway. Miner Electrolyte Metab 16:94–100Google Scholar
  16. Brown NP, Leroy C, Sander C (1998) MView: a web-compatible database search or multiple alignment viewer. Bioinformatics 14.4:380–381CrossRefGoogle Scholar
  17. Budick S, O’Malley D (2000) Locomotor repertoire of the larval zebrafish: swimming, turning and prey capture. J ExpBiol 203:2565–2579Google Scholar
  18. Cao Y, Park A, Sun Z (2010) Intraflagellar transport proteins are essential for cilia formation and for planar cell polarity. J Am Soc Nephrol 21:1326–1333CrossRefGoogle Scholar
  19. Castro A, Becerra M, Manso MJ, Anadón R (2006) Calretinin immunoreactivity in the brain of the zebrafish. Danio rerio: distribution and comparison with some neuropeptides and neurotransmitter-synthesizing enzymes. II. Midbrain, hindbrain, and rostral spinal cord. J Comp Neurol 494:792–814CrossRefGoogle Scholar
  20. Chard P, Bleakman D, Christakos S, Fullmer C, Miller R (1993) Calcium buffering properties of calbindin D28k and parvalbumin in rat sensory neurons. J Physiol 472:341–357CrossRefGoogle Scholar
  21. Christakos S, Friedlander E, Frandsen B, Norman (1979) A Studies on the mode of action of calciferol. XIII. Development of a radioimmunoassay for vitamin D-dependent chick intestinal calcium-binding protein and tissue distribution. J Endocrinol 104:1495–1503CrossRefGoogle Scholar
  22. Colovos C, Yeates T (1993) Verification of protein structures: patterns of non-bonded atomic interactions. Protein Sci 2:1511–1519CrossRefGoogle Scholar
  23. Deshpande K, Jadhao A (2015) Calcium binding protein calretinin (29kD) localization in the forebrain of the cichlid fish: an immunohistochemical study. Gen Comp Endocrinol 220:93–97CrossRefGoogle Scholar
  24. Drummond I et al (1998) Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function. Development 125:4655–4667Google Scholar
  25. Eckfeldt C et al (2005) Functional analysis of human hematopoietic stem cell gene expression using zebrafish. PLoS Biol 3(8):e254CrossRefGoogle Scholar
  26. Essner JJ, Amack JD, Nyholm MK, Harris EB, Yost HJ (2005) Kupffers vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut. Development 132:1247–1260CrossRefGoogle Scholar
  27. Fishman M (1999) Zebrafish genetics: the enigma of arrival. Proc Natl Acad Sci USA 96:10554–10556CrossRefGoogle Scholar
  28. Heizmann C, Hunzlker W (1991) Intracellular calcium-binding proteins: more sites than insights. Trends Biochem Sci 16:98–103CrossRefGoogle Scholar
  29. Hermanson S, Davidson AE, Sivasubbu S, Balciunas D, Ekker SC (2004) Sleeping beauty transposon for efficient gene delivery. Methods Cell Biol 349–362Google Scholar
  30. Hyatt T, Ekker S (1999) Vectors and techniques for ectopic gene expression in zebrafish. Methods Cell Biol 59:117–126CrossRefGoogle Scholar
  31. Jadhao A, Deshpande K (2014) Sexually dimorphic distribution of calcium-binding protein, calretinin in the preoptic area of the freshwater catfish, Clarias batrachus. Neurosci Lett 579:86–91CrossRefGoogle Scholar
  32. Jadhao A, Malz C (2007) Localization of calcium binding protein (Calretinin, 29 kD) in the brain and pituitary gland of the teleost fish: an Immunohistochemical study. Neurosci Res 59:265–276CrossRefGoogle Scholar
  33. Jaillon O et al (2004) Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature 431:946–957CrossRefGoogle Scholar
  34. Jande S, Maler L, Lawson D (1981a) Immunohistochemical mapping of vitamin D-dependent calcium binding protein in brain. Nature 294:765–767CrossRefGoogle Scholar
  35. Jande S, Toinal S, Lawson D (1981b) Immunohistochemical localization of vitamin D-dependent calcium-binding protein in duodenum, kidney, uterus and cerebellum of chickens. Histochemistry 71:99–116CrossRefGoogle Scholar
  36. Karlsson J, Von Hofsten J, Olsson P (2001) Generating transparent zebrafish: a refined method to improve detection of gene expression during embryonic development. Mar Biotechnol 3:522–527CrossRefGoogle Scholar
  37. Kimmel CB, Ballard W, Kimmel S, Ullmann B, Schilling T (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203:253–310CrossRefGoogle Scholar
  38. Kramer-Zucker AG, Olale F, Haycraft CJ, Yoder BK, Schier AF, Drummond IA (2005) Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer’s vesicle is required for normal organogenesis. Development 132:1907–1921CrossRefGoogle Scholar
  39. Kuntal BK, Aparoy P, Reddanna P (2010) EasyModeller: a graphical interface to MODELLER. BMC Res Notes 3:226–230CrossRefGoogle Scholar
  40. Lan C, Laurenson S, Copp BR, Cattin PM, Love DR (2007) Whole organism approaches to chemical genomics: the promising role of zebrafish (Danio rerio). Expert Opin Drug Discov 2:1389–1401CrossRefGoogle Scholar
  41. Lee JE (1997) Basic helix–loop–helix genes in neural development. Curr Opin Neurobiol 7:13–20CrossRefGoogle Scholar
  42. Letunic I, Bork P (2016) Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res 44:242–245CrossRefGoogle Scholar
  43. Levanti MB, Montalbano G, Laurà R, Ciriaco E, Cobo T, García-Suarez O, Germanà A, Vega JA (2007) Calretinin in the peripheral nervous system of the adult zebrafish. J Anat 212:67–71CrossRefGoogle Scholar
  44. Lin YF, Cheng CW, Shih CS, Hwang JK, Yu CS, Lu CH (2016) MIB: metal ion-binding site prediction and docking server. J Chem Inf Model 56(12):2287–2291CrossRefGoogle Scholar
  45. Lledo PM, Somasundaram B, Morton AJ, Emson PC, Mason WT (1992) Stable transfection of calbindin-D28k into the GH3 cell line alters calcium currents and intracellular calcium homeostasis. Neuron 9:943–954CrossRefGoogle Scholar
  46. Lovell S, Davis I, Arendall W, de Bakker P, Word J, Prisant M, Richardson J, Richardson D (2003) Structure validation by C-alphageometry: phi, psi and C-beta deviation. Proteins 50:437–450CrossRefGoogle Scholar
  47. Lun K, Brand MA (1998) Series of no isthmus (noi) alleles of the zebrafish pax2.1 gene reveals multiple signaling events in development of the midbrain-hindbrain boundary. Development 125:3049–3062Google Scholar
  48. Lüthy R, Bowie JU, Eisenberg D (1992) Assessment of protein models with three-dimensional profiles. Nature 5:83–85CrossRefGoogle Scholar
  49. Moews P, Kretsinger R (1975) Refinement of the structure of carp muscle calcium-binding parvalbumin by model building and difference Fourier analysis. J Mol Biol 91:201–225CrossRefGoogle Scholar
  50. Morcos P (2007) Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos. Biochem Biophys Res Commun 358:521–527CrossRefGoogle Scholar
  51. Nasevicius A, Ekker S (2000) Effective targeted gene ‘knockdown’ in zebrafish. Nat Genet 26:216–220CrossRefGoogle Scholar
  52. Nemere I, Leathers V, Thompson B, Luben R, Norman A (1991) Redistribution of calbindin-D28k in chick intestine in response to calcium transport. ‎J Endocrinol 129:2972–2984CrossRefGoogle Scholar
  53. Parmentier M, Passage E, Vassart G, Mattei MG (1991) The human calbindin D28k (CALB1) and calretinin (CALB2) genes are located at 8q21. 3 → q22.1 and 16q22 → q23, respectively, suggesting a common duplication with the carbonic anhydrase isozyme loci. Cytogenet Genome Res 57(1):41–43CrossRefGoogle Scholar
  54. Patowary A et al (2013) A sequence-based variation map of zebrafish. Zebrafish 10:15–20CrossRefGoogle Scholar
  55. Persechini A, Moncrief N, Kretsinger R (1989) The EF-hand family of calcium-modulated proteins. Trends Neurosci 12:462–467CrossRefGoogle Scholar
  56. Pickart M et al (2006) Genome-wide reverse genetics framework to identify novel functions of the vertebrate secretome. PLoS One 1(1):e104CrossRefGoogle Scholar
  57. Postlethwait J et al (1998) Vertebrate genome evolution and the zebrafish gene map. Nat Genet 18:345–349CrossRefGoogle Scholar
  58. Prosser S, Morrison C (2015) Centrin2 regulates CP110 removal in primary cilium formation. J Cell Biol 208:693–701CrossRefGoogle Scholar
  59. Ravanelli A, Klingensmith J (2011) The actin nucleator Cordon-bleu is required for development of motile cilia in zebrafish. Dev Biol 350:101–111CrossRefGoogle Scholar
  60. Reifers F, Böhli H, Walsh EC, Crossley PH, Stainier DY, Brand M (1998) Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis. Development 125:2381–2395Google Scholar
  61. Roberts W (1993) Spatial calcium buffering in saccular hair cells. Nature 363:74–76CrossRefGoogle Scholar
  62. Rogers J (1987) Calretinin: a gene for a novel calcium-binding protein expressed principally in neurons. J Cell Biol 105:1343–1353CrossRefGoogle Scholar
  63. Saint-Amant L, Drapeau P (1998) Time course of the development of motor behaviors in the zebrafish embryo. J Neurobiol 37:622–632CrossRefGoogle Scholar
  64. Schurmans S et al (1997) Impaired LTP induction in the dentate gyrus of calretinin deficient mice. Proc Natl Acad Sci USA 94:10415–10420CrossRefGoogle Scholar
  65. Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31:3381–3385CrossRefGoogle Scholar
  66. Sievers F, Wilm A, Dineen D et al (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539CrossRefGoogle Scholar
  67. Summerton J (1999) Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim Biophys Acta 1489:141–158CrossRefGoogle Scholar
  68. Sun Z et al (2004a) A genetic screen in zebrafish identifies cilia genes as a principal cause of cystic kidney. Development 131:4085–4093CrossRefGoogle Scholar
  69. Sun Z, Amsterdam A, Pazour GJ, Cole DG, Miller MS, Hopkins N (2004b) A genetic screen in zebrafish identifies cilia genes as a principal cause of cystic kidney. Development 131:4085–4093CrossRefGoogle Scholar
  70. Szklarczyk D et al (2015) STRING v10: protein–protein interaction networks, integrated over the tree of life. Nucleic Acids Res 43:447–452CrossRefGoogle Scholar
  71. UniProt Consortium (2016) UniProt: the universal protein knowledgebase. Nucleic Acids Res 45.D1: D158–D169Google Scholar
  72. Wallace AC, Roman AL, Janet MT (1995) LIGPLOT: a program to generate schematic diagrams of protein–ligand interactions. Protein Eng Des Sel 8.2:127–134CrossRefGoogle Scholar
  73. Webb B, Andrej S (2014) Protein structure modeling with MODELLER. Protein structure prediction. Humana Press, Totowa, pp 1–15Google Scholar
  74. Westerfield M (2000) The Zebrafish Book. A guide for the laboratory use of zebrafish (Danio rerio). University of Oregon Press, EugeneGoogle Scholar
  75. Westerfield M (2007) The zebrafish Book: a guide for the laboratory use of zebrafish (Danio rerio). Printed by the University of Oregon Press, EugeneGoogle Scholar
  76. Yang J, Roy A, Zhang Y (2013a) Protein–ligand binding site recognition using complementary binding-specific substructure comparison and sequence profile alignment. Bioinformatics 29:2588–2595CrossRefGoogle Scholar
  77. Yang J, Roy A, Zhang Y (2013b) BioLiP: a semi-manually curated database for biologically relevant ligand–protein interactions. Nucleic Acids Res 41:1096–1103CrossRefGoogle Scholar
  78. Yokoi H, Yan YL, Miller MR, BreMiller RA, Catchen JM, Johnson EA, Postlethwait JH (2009) Expression profiling of zebrafish sox9 mutants reveals that Sox9 is required for retinal differentiation. Dev Biol 329:1–15CrossRefGoogle Scholar
  79. Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9:40CrossRefGoogle Scholar
  80. Zündorf G, Georg R (2011) Calcium dysregulation and homeostasis of neural calcium in the molecular mechanisms of neurodegenerative diseases provide multiple targets for neuroprotection. Antioxid Redox Signal 14.7:1275–1288CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Rahul C. Bhoyar
    • 1
  • Arun G. Jadhao
    • 1
    Email author
  • Ankit Sabharwal
    • 2
  • Gyan Ranjan
    • 4
  • Sridhar Sivasubbu
    • 2
  • Claudia Pinelli
    • 3
  1. 1.Department of ZoologyRTM Nagpur UniversityNagpurIndia
  2. 2.Zebrafish Functional Genomics, CSIR Institute of Genomics and Integrative BiologyNew DelhiIndia
  3. 3.Department of Environmental, Biological and Pharmaceutical Sciences and TechnologiesUniversity of Campania “Luigi Vanvitelli”CasertaItaly
  4. 4.Department of Genetic EngineeringKattankulathur Campus, SRM Institute of Science and TechnologyChennaiIndia

Personalised recommendations