SYNERGISTIC ACTION OF BORON WITH VITAMIN D3 ON THE EARLY-STAGE SKELETAL DEVELOPMENT IN ZEBRAFISH LARVAE

Introduction

Osteogenesis is a process which can be modulated by several factors including macro and micronutrients supplementation. Among the various proven and studied micronutrients, boron (B) has an important role in the maintenance and development of bone [1–3]. Bone related diseases are a common problem today and vitamin D deficiency is one of the main reasons for weak bones. Some earlier studies showed that the boron supplementation alleviates the problems associated with vitamin D3 deficiency such as disruptions in the mineral metabolism [4]. Using zebrafish as a model, the effect of boron supplementation at two selected concentrations and its synergistic effects with vitamin D3 on skeletal development were analysed at a transcriptomic level defining the pathways modulated in the treated groups. The analysed data opens to greater understanding of the actions of these compounds to improve skeletal health which is either useful for aquaculture or biomedicine.

Materials and Methods

Zebrafish embryos were collected and divided into six groups each in triplicates- control group with ethanol (C), Vitamin D3 (VD), First concentration of boron (B10), Boron 10 with vitamin D3 (Sigma) (B10VD), Second concentration of boron (B100) and Boron100 vitamin D3 (B100VD). Boric acid (Sigma) was used to make the required concentrations of boron groups. At 7 DPH, larvae were sampled for RNA extraction and sequenced on Illumina platform to generate 150 x 2 paired end (PE) reads. ~30 million PE reads were generated across each experiment group in triplicates. Quality trimmed reads were then mapped to the reference genome assembly using STAR aligner. Read counts for the protein coding genes were retrieved using featureCounts. Differential gene expression analysis between different groups was performed using deseq2 package in R. Gene Ontology and KEGG pathway analysis was performed using clusterProfiler package in R. Plotting was done using ggplot package in R.

Results and Discussion

RNA-seq analysis identified several thousands of genes to be differentially expressed in at least one of the comparison groups (Fig1a). Clustering the data based on expression pattern revealed three different clusters (Fig1b) with one cluster clearly enriched for GO and KEGG pathways related to skeletal development. The outcomes revealed a clear evidence for boron and Vitamin D synergy contributing to enhanced bone physiology. Most affected pathway was the MAPK where many genes were seen to be highly expressed in the synergy groups than vitamin D. The calcineurin, denoted by the ppp3cca gene, which is a part multiple bone related pathways such as MAPK, WNT and Ca signalling, was seen upregulated in the B10VD synergy group and are known to be expressed in osteoblasts [5]. In the KEGG anlaysis of the TGF-β–BMP pathway, Smad4 was significantly upregulated in the B10VD group and Smad family is associated with BMP activation of genes involved in mineralisation and osteoblast differentiation [9]. In brief, the synergistic effect of boron with vitamin D3 was clearly evidenced in the skeletal pathways by the transcriptome study using zebrafish larvae model. This information can be useful for aquaculture nutrition to enrich the vitamin D3 effect by the additional supplementation of boron to enhance the ossification and alleviate the vitamin D deficiency pathologies in the cultured fishes.

References

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