Investigating the role of primary cilia

Investigating the role of primary cilia in cranial stem/progenitor cell development and craniosynostosis

Background:
Premature fusion of the cranial sutures (craniosynostosis), which affects approx. 1 in 2500 children, can restrict brain growth and often requires surgical correction. Understanding the molecular and cellular mechanisms causing craniosynostosis may lead to new therapies which will improve outcomes following surgery. The cranial sutures, which represent the sites of bone growth within the skull, are populated by two types of stem cells, migratory neural crest (NCSCs) and mesenchymal (MSCs) stem/progenitor cells [1]. Abnormal migration and/or differentiation of these stem cells causes craniosynostosis. Our lab has identified mutations in several genes causing this condition [2,3], including the IFT80 gene which is mutated in Jeune syndrome (JS). IFT80 is essential for the formation/function of primary cilia which are present on the surface of differentiating NCSCs/MSCs where they are postulated to alter stem cell differentiation programmes by initiating calcium signalling in response to pressure. Using gene-editing we have generated/characterised exact mouse models of JS carrying either p.L549del, p.A701P or p.H553X mutations, and these animals exhibit craniosynostosis.

Aims/Objectives and Methods:
Aim 1: To define the role of primary cilia in cranial suture development
Methods - In our Ift80 mouse models, microCT scanning will be used monitor the 3D structure of the top of the skull, including bone mineral density and the width of the cranial sutures at regular intervals from a few days prior to birth until several weeks after birth. The same skulls will be subject to Alcian blue/Alizarin red staining to monitor cartilage and mineralised bone, and immunohistochemistry for markers of cell proliferation, apoptosis, differentiation and cilia structure. We will also investigate how cilia regulate the transcriptional regulatory network within these systems by RNA-sequencing using material from dissected mutant and wild-type calvaria. Differentially expressed genes will be validated by qRT-PCR and in situ hybridisation. 

Aim 2: To determine how cilia influence cranial stem/progenitor cell development
Methods - We will investigate stem/progenitor cell development in several complementary ways. 1) Primary cells will be cultured from Ift80 mutant and wild-type calvaria and markers of MSCs (including Gli1, ref. 4], pre-osteoblasts and osteoblasts will be monitored by qRT-PCR. These cells are known to differentiate over 14 days in culture, and we will use this system to investigate how different Ift80 mutations influence this process. 2) External pressure is known to activate calcium signalling, resulting in transcription of differentiation factors by activation of NFAT transcription factors in pre-osteoblasts, and this requires the polycystins present within primary cilia [5]. We will investigate the requirement for cilia in this process in the skull by subjecting primary pre-osteoblasts from wild-type and mutant mice to cyclic mechanical strain. We will monitor: calcium signalling using fluorescent indicators coupled with confocal and super-resolution 3D structural illumination (SIM) microscopy; cilia formation; osteoblast differentiation using qPCR for markers of MSCs (Twist, Fgfr2), pre-osteoblasts and progenitors (Runx2, osterix, ALP, collagen I) and osteoblasts (osteocalcin). 3) Ift80 mutant mice will be crossed to Wnt1-Cre/R26R mice to label NCSCs and the distribution of NCSC-derivatives in the skull will be monitored by X-gal staining.

Aim 3: To investigate a cilia-calcium signaling axis in cranial stem cells and targeting of it for therapy
Methods - We have discovered a small molecule inhibitor of calcium signaling that reduces cranial-MSC differentiation in vitro and prevents craniosynostosis in a mouse model (Figure 1) [1]. To investigate this, we will test the drug in parallel to the in vitro experiments listed in Aims 1 & 2. We will also test the effect of daily intraperitoneal injections of the drug from E15.5 on skull development in Ift80 mutant mice, using microCT scanning to evaluate the epistatic interaction at E18.5, during the process of active ossification, and at 7 weeks after birth, when the cranial sutures have fused in craniosynostosis mouse models.

Timeline:
MicroCT, histology (Aim 1) - Year 1
Confirmation of transcriptional changes in cranial suture development (Aim 1) – during Year 2
Primary cell culture experiments (Aim 2-1, 2-2) – Year 2-3
Wnt1-Cre/R26R mouse breeding and experiment (Aim 2-3) – Year 1
Skull repair experiment (Aim 3) – Year 3

Figure 1. 3D microCT scanning and bone mineral density heat map of E18.5 mice injected with drug for 3 days, and/or carrying the Crouzon syndrome mutation in Fgfr2. Arrows indicate the coronal suture which is obliterated in mutants. The circle shows the midline sagittal suture showing that the drug reduces bone mineralisation in mutants. Bar chart shows sagittal suture measurements in wild-type and mutant mice treated with drug or vehicle alone.

References: (*denotes publications from the host lab):
*1) Seda et al. An FDA-Approved Drug Screen for Compounds Influencing Craniofacial Skeletal Development and Craniosynostosis. Mol Syndromol. 10:98-114.
*2) Jenkins et al. RAB23 mutations in Carpenter syndrome imply an unexpected role for hedgehog signaling in cranial-suture development and obesity. Am J Hum Genet. 80:1162-70.
*3) Beales et al. IFT80, which encodes a conserved intraflagellar transport protein, is mutated in Jeune asphyxiating thoracic dystrophy. Nat Genet. 39:727-9.
4) Zhao H, Feng J, Ho TV, Grimes W, Urata M, Chai Y. The suture provides a niche for mesenchymal stem cells of craniofacial bones. Nat Cell Biol. 17:386-96.
5) Dalagiorgou et al. Mechanical stimulation of polycystin-1 induces human osteoblastic gene expression via potentiation of the calcineurin/NFAT signaling axis. Cell Mol Life Sci.  70:167-80.