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

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Abstract

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.

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Abbreviations

Calb2a:

Calbindin 2a

Calb2b:

Calbindin 2b

CALB2:

Calbindin 2

CaBPs:

Calcium-binding proteins

PV:

Parvalbumin

MO:

Morpholino oligonucleotide

NIC:

Non-injected control

CNS:

Central nervous system

hpf:

Hours post fertilization

dpf:

Day post fertilization

MHB:

Midbrain-hindbrain boundary

Tg:

Tegmentum

Hb:

Hindbrain

nL:

Nanolitre

mM:

Millimolar

mm:

Millimetre

pg:

Picogram

PTU:

N-phenylthiourea

pdb:

Protein database

BLAST:

Basic Local Alignment Search Tool

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Acknowledgements

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.

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Authors and Affiliations

  1. Department of Zoology, RTM Nagpur University, Nagpur, 440033, India

    Rahul C. Bhoyar & Arun G. Jadhao

  2. Zebrafish Functional Genomics, CSIR Institute of Genomics and Integrative Biology, New Delhi, India

    Ankit Sabharwal & Sridhar Sivasubbu

  3. Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania “Luigi Vanvitelli”, 81100, Caserta, Italy

    Claudia Pinelli

  4. Department of Genetic Engineering, Kattankulathur Campus, SRM Institute of Science and Technology, Chennai, Tamil Nadu, 603203, India

    Gyan Ranjan

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  1. Rahul C. Bhoyar
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  2. Arun G. Jadhao
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  3. Ankit Sabharwal
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  4. Gyan Ranjan
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  5. Sridhar Sivasubbu
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  6. Claudia Pinelli
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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.

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Correspondence to Arun G. Jadhao.

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429_2018_1797_MOESM1_ESM.pdf

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)

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Bhoyar, R.C., Jadhao, A.G., Sabharwal, A. et al. Knockdown of calcium-binding calb2a and calb2b genes indicates the key regulator of the early development of the zebrafish, Danio rerio. Brain Struct Funct 224, 627–642 (2019). https://doi.org/10.1007/s00429-018-1797-8

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