Work Description

Date: January 5, 2020

Dataset Title: FGFR2-C342Y/+ and FGFR2+/+ Treated with/without Tissue Nonspecific AlkalinePhosphatase (TNAP) Mouse Skulls: BalbC strain 4 weeks postnatal

Dataset Creators: H.K. Nam, I. Vesela, S.D. Schutte and N.E. Hatch

Funding: NIH/NIDCR R01DE025827 (N.E.H.)

Key Points:
-We treated neonatal mice carrying the FGFR2-C342Y mutation causal for Crouzon craniosynostosis with lentivirus encoding TNAP (tissue nonspecific alkaline phosphatase) to determine if treatment with exogenous TNAP would diminish craniosynostosis severity
- Treatment with TNAP significantly diminished craniosynostosis and malocclusion in FGFR2C342Y/+ mice on the C57BL/6 but not the BALB/c genetic background
- These results indicate that post-natal recombinant TNAP enzyme therapy can diminish craniosynostosis severity in the FGFR2C342Y/+ mouse model of Crouzon syndrome, and that effects of exogenous TNAP are genetic background dependent.
- The results also indicate that cellular mechanisms leading to craniosynostosis in FGFR2C342Y/+ mice are both independent and dependent upon TNAP.

Research Overview:
Craniosynostosis is the premature fusion of cranial bones. The goal of this study was to determine if delivery of recombinant tissue nonspecific alkaline phosphatase (TNAP) could prevent or diminish the severity of craniosynostosis in a C57BL/6 FGFR2C342Y/+ model of neonatal onset craniosynostosis or a BALB/c FGFR2C342Y/+ model of postnatal onset craniosynostosis. Mice were injected with a lentivirus encoding a mineral targeted form of TNAP immediately after birth. Cranial bone fusion as well as cranial bone volume, mineral content and density were assessed by micro computed tomography. Craniofacial shape was measured with calipers. Alkaline phosphatase, alanine amino transferase (ALT) and aspartate amino transferase (AST) activity levels were measured in serum. Neonatal delivery of TNAP diminished craniosynostosis severity from 94% suture obliteration in vehicle treated mice to 67% suture obliteration in treated mice, p<0.02) and the incidence of malocclusion from 82.4% to 34.7% (p<0.03), with no effect on cranial bone in C57BL/6 FGFR2C342Y/+ mice. In contrast, treatment with TNAP improved cranial bone volume (p< 0.01), density (p< 0.01) and mineral content (p< 0.01) but had no effect on craniosynostosis or malocclusion in BALB/c FGFR2C342Y/+ mice. These results indicate that post-natal recombinant TNAP enzyme therapy diminishes craniosynostosis severity in the C57BL/6 FGFR2C342Y/+ neonatal onset mouse model of Crouzon syndrome, and that effects of exogenous TNAP are genetic background dependent.

Methodology:
TNAP Lentivirus
Recombinant mineral-targeted TNAP lentivirus expresses a mineral-targeted protein that is composed of soluble human TNAP enzyme fused to the constant region of human IgG1 and a C-terminal deca-aspartate motif to confer targeting to hydroxyapatite. Production and titer of the lentivirus for this study was performed by the University of Michigan Vector Core.

Animal Procedures
FGFR2C342Y/+mice were backcrossed onto BALB/c and C57BL/6 strains for at least fifteen generations prior to experiments. BALB/c FGFR2C342Y/+mice have a more moderate form of Crouzon syndrome with craniosynostosis in the form of point fusions across the coronal suture first apparent between three and four weeks after birth. C57BL/6 mice have a more severe form of Crouzon syndrome with craniosynostosis in the form of suture obliteration first apparent in neonatal mice. Genotyping was performed by PCR. Treated mice were injected with 1.0 x 107 transforming units lentivirus or an equivalent volume of phosphate buffered saline via the jugular vein after birth.

Micro Computed Tomography
Whole skulls were scanned at an 18 µm isotropic voxel resolution using the eXplore Locus SP micro computed tomography imaging system (GE Healthcare Pre-Clinical Imaging, London, ON, Canada). Regions of interest (ROI’s) for parietal and frontal bones were established as 0.5 mm in length, 0.5 mm in width, and depth equivalent to thickness of bone. Density, volume and mineral content of cranial bones from mice were measured using previously established methods using Microview version 2.2 software (GE Healthcare Pre-Clinical Imaging, London, ON).

Instrument and/or Software specifications:
eXplore Locus SP micro computed tomography imaging system (GE Healthcare Pre-Clinical Imaging, London, ON, Canada)
Microview version 2.2 software (GE Healthcare Pre-Clinical Imaging, London, ON)
Dolphin Imaging 11.0 software (Dolphin Imaging and Management Solutions, Chatsworth, CA)

Files contained here:

Genotype Treated Mouse ID
WT PBS C1 1
WT PBS C2 2
WT PBS C3 5
WT PBS C3 6
WT PBS C4 3
WT PBS C5 1
WT PBS C1 2
WT PBS C1 3
WT PBS C2 3
WT PBS C4 4
CZ PBS C2 1
CZ PBS C3 1
CZ PBS C3 2
CZ PBS C3 3
CZ PBS C3 4
CZ PBS C4 1
CZ PBS C4 2
CZ PBS C5 2
CZ PBS C5 3
CZ PBS C5 4
CZ PBS C1 4
CZ PBS C5 6
CZ PBS C5 7
WT TNAP T1 2
WT TNAP T2 4
WT TNAP T3 1
WT TNAP T4 2
WT TNAP T5 1
WT TNAP T5 2
WT TNAP T5 4
WT TNAP T6 3
WT TNAP T6 6
WT TNAP T6 7
WT TNAP T7 1
WT TNAP T7 2
WT TNAP T8 1
WT TNAP T8 2
WT TNAP T8 4
WT TNAP T8 6
WT TNAP T8 7
WT TNAP T1 3
WT TNAP T2 1
WT TNAP T2 2
WT TNAP T2 3
WT TNAP T3 2
WT TNAP T3 4
WT TNAP T4 3
WT TNAP T4 4
WT TNAP T5 5
WT TNAP T6 4
WT TNAP T6 5
WT TNAP T6 8
WT TNAP T6 9
CZ TNAP T1 4
CZ TNAP T4 1
CZ TNAP T5 3
CZ TNAP T7 4
CZ TNAP T7 5
CZ TNAP T8 3
CZ TNAP T8 5
CZ TNAP T3 5
CZ TNAP T4 5
CZ TNAP T4 6
CZ TNAP T6 1
CZ TNAP T6 2
CZ TNAP T7 3
CZ TNAP T8 8
CZ TNAP T8 9

3D micro CT files in .vff format were created and can be accessed using publicly available MicroView software (http://www.parallax-innovations.com/microview.html). The MicroView viewer is open source, and the program can be used to convert .vff to other file types, including DICOM files.

Related publications:
Viral delivery of tissue nonspecific alkaline phosphatase diminishes craniosynostosis in one of two FGFR2C342Y/+ mouse models of Crouzon syndrome.

Use and Access:
This data set is made available under an Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).

To Cite Data:
Hatch, N. FGFR2-C342Y/+ and FGFR2+/+ Treated with/without Tissue Nonspecific AlkalinePhosphatase (TNAP) Mouse Skulls: BalbC strain 4 weeks postnatal [Data set]. University of Michigan - Deep Blue. https://doi.org/10.7302/hmey-ab78