BMPR-IB gene disruption causes severe limb deformities in pigs
摘要: 在之前的研究中，我们原本计划通过CRISPR/Cas9编辑技术在猪的BMPR-IB基因中产生g.A746G的突变，然而却意外地获得了BMPR-IB纯合敲除仔猪（BMPR-IB-/-）和携带A746G突变的杂合子敲除仔猪（BMPR-IB-/746G）。PCR检测及测序结果显示BMPR-IB基因区域中发生了复杂的基因组重排。所有BMPR-IB-/-和BMPR-IB-/746G仔猪都无法正常站立和行走。解剖学观察发现，这些仔猪均呈现出严重的四肢骨骼发育异常，其中前肢的前臂骨（尺骨、桡骨）扭曲及截短，腕骨、掌骨和指骨排列紊乱；相比前肢，后肢表现出更严重的畸形，包括腓侧半肢畸形、跗骨增大和脚趾关节骨排列紊乱。与BMPR-IB-/746G个体相比，BMPR-IB-/-仔猪的四肢骨骼畸形更为严重。通过蛋白质组学分析，在杂合子敲除仔猪（BMPR-IB-/746G）和对照组仔猪（WT）的后肢腓骨中鉴别到139个差异表达蛋白（DEPs），大多数DEPs是已知的参与骨骼或胚胎发育、TGF-β通路或肿瘤进展的重要因子；基因本体（GO）和蛋白质结构域富集分析也显示出这些通路上的变化。在排列前50的DEPs中，有一大部分蛋白如C1QA、MYO1H、SRSF1、P3H1、GJA1、TCOF1、RBM10、SPP2、MMP13及PHAX，均已被证明与骨骼发育显著相关。该研究为解析BMPR-IB在哺乳动物肢体发育中的作用提供了新的认识。Abstract: In an attempt to generate g.A746G substitution in the BMPR-IB gene, we unexpectedly obtained BMPR-IB homozygous knockout piglets (BMPR-IB-/-) and heterogeneous knockout piglets with one copy of the A746G mutation (BMPR-IB-/746G) via CRISPR/Cas9 editing. Polymerase chain reaction (PCR) and sequencing revealed complex genomic rearrangements in the target region. All BMPR-IB-disrupted piglets showed an inability to stand and walk normally. Both BMPR-IB-/- and BMPR-IB-/746G piglets exhibited severe skeletal dysplasia characterized by distorted and truncated forearms (ulna, radius) and disordered carpal, metacarpal, and phalangeal bones in the forelimbs. The piglets displayed more severe deformities in the hindlimbs by visual inspection, including fibular hemimelia, enlarged tarsal bone, and disordered toe joint bones. Limb deformities were more profound in BMPR-IB-/- piglets than in the BMPR-IB-/746G piglets. Proteomic analysis identified 139 differentially expressed proteins (DEPs) in the hindlimb fibula of BMPR-IB-/746G piglets compared to the wild-type (WT) controls. Most DEPs are involved in skeletal or embryonic development and/or the TGF-β pathway and tumor progression. Gene Ontology (GO) and protein domain enrichment analysis suggested alterations in these processes. Of the top 50 DEPs, a large proportion, e.g., C1QA, MYO1H, SRSF1, P3H1, GJA1, TCOF1, RBM10, SPP2, MMP13, and PHAX, were significantly associated with skeletal development. Our study provides novel findings on the role of BMPR-IB in mammalian limb development.
Figure 1. Generation of BMPR-IB-modified PFFs and piglets
A: Schematic of strategy used to generate BMPR-IB-modified PFFs via linear double-stranded DNA-mediated homology-directed repair. Q, glutamine; R, arginine. B: Long-range PCR detection of BMPR-IB-deleted mutations in cloned piglets and PFF colonies. Lane 15 is a negative control (water) and M is a DNA marker. C: Sequencing chromatograms showing BMPR-IB A746G mutation in cloned piglets. Mutated nucleotide is marked by red box. D: Sequencing chromatograms showing BMPR-IB KO mutations in cloned piglets. Target site is marked by a red box. KO, knockout. E: Genotypes of newborn piglets. WT, wild-type; BMPR-IB-/746G, harboring 746G and 1 365 bp del in trans; BMPR-IB-/-, harboring 746A and 2 bp del/2 431 bp del. F: Genome rearrangements of BMPR-IB gene in BMPR-IB-/746G and BMPR-IB-/- piglets. Deleted regions are marked in red.
Figure 2. Phenotypic characterization of BMPR-IB-disrupted piglets
A: Whole-body photographs of symptomatic BMPR-IB-disrupted piglets (BMPR-IB-/746G and BMPR-IB-/-) and WT piglets. B: Radiographs of forelimb and hindlimb in piglets of three different genotypes. Absence of fibula is noted in mutant piglets (yellow arrow). Red arrow indicates third proximal phalanx (os compedale). One BMPR-IB-/-, two BMPR-IB-/746G, and two WT-cloned piglets were used for radiography. C: Anatomical views of underdeveloped skeleton of forelimb in mutant piglet compared to WT piglet. D: Anatomical views of underdeveloped skeleton of hindlimb of mutant piglet compared to WT piglet. Partial or complete absence of fibula (yellow arrow) can be observed in BMPR-IB-/746G and BMPR-IB-/- piglets. Third proximal phalanx (os compedale) fell off (red arrow) in BMPR-IB-/746G piglets. White bars: 2 cm.
Figure 3. Micro-CT analyses of forelimb bones of piglets
A: High-resolution micro-CT scans of forelimb skeletons of WT, BMPR-IB-/746G, and BMPR-IB-/- piglets. B: 3D and 2D gray-scale images of radius of three different genotype piglets. C: Quantitative results of trabecular and cortical bone at radius distal end. Data are means±SD (WT, n=4; BMPR-IB-/746G, n=4; BMPR-IB-/-, n=2). ns: P>0.05; *: P<0.05; **: P<0.01. White bars: 1 cm. BV/TV, trabecular bone volume fraction; BS/TV, trabecular bone surface density; Tb.Sp, trabecular spacing; Tb.N, trabecular number; Tb.BMD, trabecular bone mineral density; Ct.BMD, cortical bone mineral density; Ct.Th, cortical thickness; Ct.ar/Tt.ar, cortical area fraction.
Figure 4. Micro-CT analyses of hindlimb bones of piglets
A: High-resolution micro-CT scans of hindlimb skeletons of WT, BMPR-IB-/746G, and BMPR-IB-/- piglets. B: 3D and 2D gray-scale images of tibia of three different genotype piglets. C: Quantitative results of trabecular and cortical bone at tibia distal end. Data are means±SD (WT, n=4; BMPR-IB-/746G, n=4; BMPR-IB-/-, n=2). ns: P>0.05, *: P<0.05; **: P<0.01. White bars: 1 cm.
Figure 5. qRT-PCR and western blot analyses of BMPR-IB expression levels
A: Relative BMPR-IB mRNA expression levels in liver, kidney, testicle, forelimb cartilage, hindlimb cartilage, forelimb ulna, and hindlimb fibula of WT, BMPR-IB-/746G, and BMPR-IB-/- piglets, determined by qRT-PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control. B: Western blots of BMPR-IB in forelimb ulna and hindlimb fibula in three BMPR-IB-/746G piglets and three WT piglets. Liver and kidney samples of one BMPR-IB-/- piglet were used as negative controls. C: Quantitative results of BMPR-IB protein using ImageJ software. β-actin was used as a control. Data are mean±SD (WT, n=3; BMPR-IB-/746G, n=3; BMPR-IB-/-, n=1). *: P<0.05; **: P<0.01. As BMPR-IB mRNA and protein expression levels were not detectable in BMPR-IB-/- piglet samples, expression level columns are not displayed in (A) and (C) in this figure.
Figure 6. DEPs in hindlimb fibula between BMPR-IB-/746G and WT groups
A: Volcano-plot distribution map. Red and blue dots denote significantly up- and down-regulated proteins, respectively. B: GO analysis of DEPs. Top 30 significantly enriched categories are shown in horizontal histogram. Horizontal axis represents –log10(P-value); vertical axis represents GO functional classification. C: Protein domain enrichment analysis of DEPs. Top 10 significantly enriched categories are shown in bubble chart. Functional classification is shown along vertical axis of bubble chart; proportion of DEPs by functional type divided by the ratio of identified proteins was log2 converted and shown along horizontal axis. Circle color indicates enriched P-value, and circle size indicates number of DEPs in functional classification.
Figure 7. Western blot analyses of DEP expression levels in hindlimb fibula between BMPR-IB-/746G and WT groups
A: Western blots of DEPs in hindlimb fibula from three BMPR-IB-/746G piglets and three WT piglets. B: Quantitative analysis results of DEPs using ImageJ software. β-actin was used as a control. Data are mean±SD (WT, n=3; BMPR-IB-/746G, n=3). *: P<0.05; **: P<0.01.
Table 1. PRM-verified DEPs in hindlimb fibula of BMPR-IB-/746G piglets compared with WT individuals
Protein accession Gene BMPR-IB-/746G/WT ratio (PRM) BMPR-IB-/746G/WT
BMPR-IB-/746G/WT ratio (LQ) F1S4R5 SRSF7 1.79 3.49E-04 1.69 Q29101 GJA1 1.52 1.45E-02 1.52 P14287 SPP1 1.93 1.52E-02 2.48 F1SV56 MMP13 1.97 1.72E-02 2.19 P09571 TF 4.38 2.19E-02 2.78 Q8HZV3 TFRC 1.66 2.31E-02 1.51 F1SM14 SPP2 3.15 2.52E-02 4.30 PRM: Parallel reaction monitoring; LQ: 4D label-free quantitative proteomic analysis.
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ZR-2021-291 Supplementary Material.pdf