1 / 24

Transcriptome atlases of the craniofacial sutures

Transcriptome atlases of the craniofacial sutures. Harm van Bakel, PhD. Suture development. Osteogenic fronts - proliferation and differentiation of preosteoblasts Suture mesenchyme - separates osteogenic fronts; later a niche for osteoblast stem cells

manualt
Download Presentation

Transcriptome atlases of the craniofacial sutures

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Transcriptome atlases of thecraniofacial sutures Harm van Bakel, PhD

  2. Suture development • Osteogenic fronts - proliferation and differentiation of preosteoblasts • Suture mesenchyme - separates osteogenic fronts; later a niche for osteoblast stem cells • Morphology varies between sutures, e.g. end-to-end vs. overlapping Zhao et al., 2015

  3. Differential gene expression and suture development Coronal Suture p f Apert syndrome Fgfr2+/S252W Mathijssen et al., 1996 P0 Mouse Human Mouse Human Holmes et al., 2009 9 months P0 f p Saethre-Chotzen syndrome Twist1+/- 1 Johnson et al., 2000 El Ghouzzi et al., 1997 Comprehensive expression profiling needed to understand suture biology

  4. 11 Sutures – Multiple ages for WT and craniosynostosisgenotypes Chris Percival

  5. Laser capture microdissection of murine suture regions Frontal Suture

  6. WT suture expression profiles Frontal 285 WT profiles 285 WT profiles

  7. Fgfr2+/S252W and Twist1+/- suture expression profiles Frontal 350 mutant profiles → 635 total

  8. Facebase bulk RNA-Seq datasets Frontal Coronal Internasal • 7 suture types • E16.5 and E18.5 for WT, Fgfr2 or Twist1 mutants • ~340 RNA-Seq libraries Interpremaxillary Intermaxillary Interpalatine Maxillary-palatine >7,000 differentially expressed genes across suture regions, development, and mutants 4 suture types, 18 individual sutures, 200 RNA-Seq profiles

  9. Co-expression network analysis Network module WT Frontal suture mesenchyme

  10. Module hierarchy of the WT frontal suture mesenchyme

  11. Network module properties in frontal suture mesenchyme Co-expression network modules → Osteogenic

  12. Module with genes encoding extracellular space proteins DS splicing Increased at E16.5 Increased at E18.5 Frontal suture mesenchyme

  13. Differential splicing analysis – coronal suture Overview of local splicing events Alternative splicing in coronal suture (FDR 0.01) Exon skip Mutually exclusive exons Alternative 3’ or 5’ Splice sites (A3/5SS) Alternative promoter Alternative 3’ exon

  14. Alternate splicing of Postn, Plod2, and Runx2 Postn Exon 17, increased skip in FR Fasciclin domain Plod2 Exon 14, increased skip in FR Nucleotide-diphospho-sugar transferase Runx2 Exon 8, increased skip in FR Runt-relatedTF domain Reference transcripts Exon skip Protein domain

  15. Dissecting suture cell populations osteogenic front osteogenic front Zhao et al., 2015 Arboleya et al. 2013

  16. Suture subpopulations in osteogenic fronts osteogenic front osteogenic front Zhao et al., 2015 WT E18.5 Immunohistochemistry: RUNX2,BCL11B Holmes et al., 2015

  17. Single-cell sequencing of 4 WT sutures at 3 ages • WT Sutures: • Coronal • Lambdoid • Frontal • Sagittal Profiled Sagittal Suture • E18.5 (as in Atlases, active formation) • P10 (skull expansion, frontal suture fusion imminent) • P28 (post-expansion, mature suture maintenance) Zimmerman et al., 1998

  18. 10X genomics chromium single-cell RNA-Seq Cells + reagents + oil Library synthesis Single cell Functionalized gel bead RT reagents in solution Partitioning oil (Courtesy of Robert Sebra, Icahn School of Medicine at Mount Sinai)

  19. Cell types in the P10 lambdoid suture Cell-type specific expression Cell type clustering Chondrocytes Pericytes Capillary endothelial cells Osteoclasts B cells T cells Macrophages (C1q+) Neutrophils Dendritic cells Mesenchyme Macrophages Osteoblasts

  20. Cell type composition differs between sutures and age CE18 – Coronal suture E18 CP10 – Coronal suture P10 LE18 – Lambdoid suture E18 LP10 – Lambdoid suture P10

  21. Cell type composition differs between sutures and age CE18 – Coronal suture E18 CP10 – Coronal suture P10 LE18 – Lambdoid suture E18 LP10 – Lambdoid suture P10 Excluding cartilage cells

  22. Lineage profiling of P10 lambdoid suture mesenchyme Mesenchymal genes Osteoblast genes Mesenchyme Osteoblast Mesenchymal cell types Top-5 genes See poster by Ana Sylvia Gonzalez-Reiche

  23. Using single-cell data to dissect LCM bulk RNA-Seq data Co-expression network modules → Osteogenic

  24. Acknowledgements Icahn School of Medicine at Mount Sinai Ethylin Wang Jabs Harm van Bakel Greg Holmes Ana Sylvia Gonzalez-Reiche Joshua Rivera Divya Kriti Na Lu Bhavana Shewale Bin Zhang Xianxiao Zhou Dalila Pinto Michael Donovan Cincinnati Children’s Medical Center Steven Potter NIH/NIDCR 5U01 DE024448

More Related