Transcriptome Analysis Elucidates the Potential Key Genes Involved in Rib Development in bmp6-Deficient Silver Carp (Hypophthalmichthys molitrix)

Abstract

Bone morphogenetic protein 6 (BMP-6) is a constituent of the TGF-β superfamily, known for its ability to stimulate bone and cartilage formation. The investigation of bmp6's involvement in the formation of intermuscular bones in fish has garnered significant attention in recent years. The rib cage is an important skeletal structure that plays a protective function for internal organs in fish. However, there has been limited research conducted on the effects of the bmp6 gene on rib development. Silver carp is one of four major fish in China, favoured for its affordability and tender muscle. Nevertheless, the presence of numerous intermuscular bones in silver carp significantly hinders the advancement of its palatability and suitability for processing. This study showcases the effective utilisation of CRISPR/Cas9 technology for the purpose of disrupting the bmp6 gene in silver carp, leading to the creation of chimeras in the P₀ generation, marking the first instance of such an achievement. The chimeras exhibited complete viability, normal appearance, and partial intermuscular bones loss, with approximately 30% of them displaying rib bifurcation or bending. Subsequently, a transcriptome analysis on ribs of P₀ chimeras and wild-type silver carp was conducted, leading to the identification of 934 genes exhibiting differential expression, of which 483 were found to be up-regulated and 451 were found to be down-regulated. The results of the KEGG analysis revealed that the "NF-kappa B signalling pathway", "Hippo signalling pathway", "osteoclast differentiation", and "haematopoietic cell lineage" exhibited enrichment and displayed a significant correlation with bone development. The up-regulated genes such as tnfα, fos, and ctgf in pathways may facilitate the proliferation and differentiation of osteoclasts, whereas the down-regulation of genes such as tgfb2 and tgfbr1 in pathways may hinder the formation and specialisation of osteoblasts, ultimately resulting in rib abnormalities. This study presents novel findings on the impact of bmp6 gene deletion on the rib development of silver carp, while simultaneously investigating the previously unexplored molecular mechanisms underlying rib defects in fish.

Keywords: Hypophthalmichthys molitrix; bmp6; rib; transcriptome analysis.

Figure 1

Knocking out bmp6 gene by CRISPR/Cas9 technology in silver carp. (A) Schematic representation of bmp6 gene structure in silver carp shows the three gRNA targets located within exon 1. The green squares represent exons, the yellow squares represent introns, and the gRNA target sequences are indicated in blue. (B) Schematic diagram illustrates three distinct instances of bmp6 mutation types observed in P₀ chimeras. WT denotes the sequence of the bmp6 gene in the wild-type silver carp. Type 1–3 signifies the frameshift mutations identified in the bmp6 gene of the P₀ chimeras.

Figure 2

Alizarin Red staining of WT and P0 chimera individuals. Scale bar = 2 mm. Chimeras with a knockout of the bmp6 gene exhibit curved and bifurcate rib phenotypes, as indicated by dashed lines and asterisks representing curved ribs, and arrows representing bifurcate ribs. (A), wild-type silver carp; (B), chimeric rib-curved silver carp; (C), chimeric rib-bifurcate silver carp. WT: wild-type silver carp; CH: P0 chimeras silver carp.

Figure 3

Identification of the DEGs in ribs between WT and CH groups. (A) Volcano plot of DEGs. Red dots indicate the up-regulated genes and blue dots indicate the down-regulated genes. (B) Heatmap representation of the DEGs clustering analysis Genes are arranged in a horizontal orientation, with each column representing a distinct sample. The colour red is employed to signify genes that are expressed at a high level, while the colour blue is utilised to indicate genes that are expressed at a low level. WT: wild-type silver carp; CH: P₀ chimeras silver carp.

Figure 4

Functional enrichment analysis of the DEGs in ribs between WT and CH groups. (A) GO enrichment analysis of DEGs The X axis represents the number of DEGs; each bar in Y axis represents one enriched term and is coloured by −log10 (p-value). (B) KEGG enrichment analysis of DEGs. The X-axis indicates the enrichment factor, and the Y-axis indicates the KEGG pathways. The larger the bubble, the more genes enriched in the pathway, and the colour of the bubble represents the p-value.

Figure 5

The heatmap illustrates the changes in gene expression within four pathways related to bone development. The average RPKM value of each gene is used to plot the heatmaps. Up-regulated genes are visually represented in red, whereas down-regulated genes are visually represented in blue. The genes marked with boxes are identified as participating in multiple pathways. WT: wild-type silver carp; CH: P₀ chimeras silver carp.

Figure 6

RT-qPCR validation of eight DEGs, including ctgf (A), fos (B), metk (C), irak4 (D), card1l (E), igh (F), got1 (G), and bhmt (H). Y-axis exhibits the relative expression level of genes. WT: wild-type silver carp; CH: P₀ chimeras silver carp. (I) A correlation analysis between the RNA-seq and qRT-PCR data. The reference line indicates the linear relationship between the results of RNA-seq and qRT-PCR. Error bars represent the standard deviation of three replicates. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate significant differences.