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An RNA-seq protocol to identify mRNA expression changes in mouse diaphyseal bone: applications in mice with bone
Ugur M Ayturk1, Christina M Jacobsen, Danos C Christodoulou
1Department of Orthopaedic Surgery, Boston Children's Hospital, Boston, MA, USA; Department of Genetics, Harvard Medical School, Boston, MA, USA.
Abstract:
Loss-of-function and certain missense mutations in the Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) significantly decrease or increase bone mass, respectively. These human skeletal phenotypes have been recapitulated in mice harboring Lrp5 knockout and knock-in mutations. We hypothesized that measuring mRNA expression in diaphyseal bone from mice with Lrp5 wild-type (Lrp5(+/+) ), knockout (Lrp5(-/-) ), and high bone mass (HBM)-causing (Lrp5(p.A214V/+) ) knock-in alleles could identify genes and pathways that regulate or are regulated by LRP5 activity. We performed RNA-seq on pairs of tibial diaphyseal bones from four 16-week-old mice with each of the aforementioned genotypes. We then evaluated different methods for controlling for contaminating nonskeletal tissue (ie, blood, bone marrow, and skeletal muscle) in our data. These methods included predigestion of diaphyseal bone with collagenase and separate transcriptional profiling of blood, skeletal muscle, and bone marrow. We found that collagenase digestion reduced contamination, but also altered gene expression in the remaining cells. In contrast, in silico filtering of the diaphyseal bone RNA-seq data for highly expressed blood, skeletal muscle, and bone marrow transcripts significantly increased the correlation between RNA-seq data from an animal's right and left tibias and from animals with the same Lrp5 genotype. We conclude that reliable and reproducible RNA-seq data can be obtained from mouse diaphyseal bone and that lack of LRP5 has a more pronounced effect on gene expression than the HBM-causing LRP5 missense mutation. We identified 84 differentially expressed protein-coding transcripts between LRP5 "sufficient" (ie, Lrp5(+/+) and Lrp5(p.A214V/+) ) and "insufficient" (Lrp5(-/-) ) diaphyseal bone, and far fewer differentially expressed genes between Lrp5(p.A214V/+) and Lrp5(+/+) diaphyseal bone.
Insights
Investigating low-density lipoprotein receptor-related protein 5 (LRP5) gene expression in mouse bone revealed that LRP5 deficiency significantly impacts gene activity. In silico filtering improves RNA-seq data reliability for studying bone mass regulation.
Area of Science:
- Genetics and Genomics
- Skeletal Biology
- Molecular Biology
Background:
- Mutations in the Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) are linked to altered bone mass in humans and mice.
- Understanding LRP5's role requires analyzing gene expression in bone tissue, but contamination from non-skeletal tissues is a challenge.
Purpose of the Study:
- To identify genes and pathways regulated by LRP5 activity using RNA sequencing (RNA-seq) in mouse diaphyseal bone.
- To evaluate methods for minimizing non-skeletal tissue contamination in bone RNA-seq data.
- To compare the effects of LRP5 loss-of-function versus a high bone mass-causing mutation on gene expression.
Main Methods:
- RNA sequencing of tibial diaphyseal bone from mice with wild-type, knockout, and high bone mass-associated LRP5 alleles.
- Evaluation of collagenase digestion versus in silico filtering to remove contaminating transcripts from blood, bone marrow, and skeletal muscle.
- Differential gene expression analysis between LRP5 genotypes.
Main Results:
- In silico filtering of RNA-seq data effectively reduced contamination and improved data reproducibility compared to collagenase digestion.
- LRP5 deficiency had a more substantial impact on gene expression than the high bone mass-causing LRP5 mutation.
- 84 differentially expressed protein-coding transcripts were identified between LRP5-sufficient and LRP5-insufficient diaphyseal bone.
Conclusions:
- Reliable and reproducible RNA-seq data can be obtained from mouse diaphyseal bone using in silico filtering methods.
- LRP5 plays a critical role in regulating gene expression within the diaphyseal bone.
- This study provides a foundation for further investigation into LRP5-mediated skeletal development and disease.
