Altered bone growth dynamics prefigure craniosynostosis in a zebrafish model of Saethre-Chotzen syndrome

  1. Camilla S Teng
  2. Man-chun Ting
  3. D'Juan T Farmer
  4. Mia Brockop
  5. Robert E Maxson  Is a corresponding author
  6. J Gage Crump  Is a corresponding author
  1. University of Southern California, United States
8 figures, 1 table and 3 additional files

Figures

Figure 1 with 3 supplements
Coronal suture loss in tcf12; twist1b mutant zebrafish.

(A) Diagrams of zebrafish, mouse, and human skulls, with neural crest contributions in turquoise and mesoderm contributions in gold. The coronal suture is at a mesoderm-mesoderm boundary in …

https://doi.org/10.7554/eLife.37024.003
Figure 1—figure supplement 1
Zebrafish TALEN mutants.

The sites of nucleotide changes induced by TALEN cleavage are shown for each mutant allele. Schematics show the predicted protein truncations caused by the frame-shift mutations, relative to the …

https://doi.org/10.7554/eLife.37024.004
Figure 1—figure supplement 2
Patent sutures in tcf12 single mutants.

Skull bones of an adult tcf12-/- fish were stained with Alizarin Red. Scale bar, 1 mm. Sections of the coronal suture (cs) and sagittal suture (ss) were stained with hematoxylin and eosin to show …

https://doi.org/10.7554/eLife.37024.005
Figure 1—figure supplement 3
Zebrafish skull phenotypes in different mutant combinations.

Dorsal views of adult zebrafish skull bones stained with Alizarin Red. We observed coronal suture loss (asterisks) in tcf12-/-; twist1b-/- mutants of any twist1a genotype (D–F). Animals lacking both …

https://doi.org/10.7554/eLife.37024.006
Loss of tcf12 partially rescues twist1a; twist1b jaw cartilage defects and viability.

(A) In situ hybridizations at 20 hpf show abnormal persistence of sox10 expression in arch ectomesenchyme (boxed region) in twist1a-/-; twist1b-/- and tcf12-/-; twist1a-/-; twist1b-/- mutants. …

https://doi.org/10.7554/eLife.37024.007
Figure 2—source data 1

Cartilage size in twist1a;twist1b mutants.

https://doi.org/10.7554/eLife.37024.008
Figure 2—source data 2

Severity of cartilage defects in combinatorial Twist1 and Tcf12 mutants.

https://doi.org/10.7554/eLife.37024.009
Figure 2—source data 3

Adult viability of combinatorial Twist1 and Tcf12 mutants.

https://doi.org/10.7554/eLife.37024.010
Expression of tcf12 and twist1b in multiple sutures of zebrafish.

(A) Schematic of the zebrafish skull depicting positions of sections (dotted lines) used for RNAscope in situ hybridizations. cs, coronal suture; ms, metopic suture; ss, sagittal suture; fb, frontal …

https://doi.org/10.7554/eLife.37024.011
Altered bone growth dynamics precede craniosynostosis in mutant zebrafish.

(A) Dorsal views of the developing skull bones in the same wild-type and mutant individuals across four developmental stages. Live fish were stained with Calcein Green at 6, 7, and 8 mm and Alizarin …

https://doi.org/10.7554/eLife.37024.012
Figure 4—source data 1

Quantification of skull bone area in mutants by 10.25 mm.

https://doi.org/10.7554/eLife.37024.013
Figure 4—source data 2

Directionality of bone growth in mutants.

https://doi.org/10.7554/eLife.37024.014
Figure 4—source data 3

Quantification of growth in coronal area.

https://doi.org/10.7554/eLife.37024.015
Figure 4—source data 4

Quantification of growth in metopic area.

https://doi.org/10.7554/eLife.37024.016
Figure 4—source data 5

Quantification of growth in sagittal area.

https://doi.org/10.7554/eLife.37024.017
Figure 5 with 2 supplements
Altered proliferation and osteoblast production at mutant zebrafish bone fronts.

(A) Dissected skullcaps were stained for BrdU (magenta) and Sp7 protein (green) at 9 mm. Top panels show maximum intensity projections of whole skull volumes, and middle panels are the same volumes …

https://doi.org/10.7554/eLife.37024.018
Figure 5—source data 1

Quantification of proliferative sp7+ osteoblasts in mutant zebrafish.

https://doi.org/10.7554/eLife.37024.021
Figure 5—source data 2

Quantification of proliferative sp7- cells in mutant zebrafish.

https://doi.org/10.7554/eLife.37024.022
Figure 5—figure supplement 1
Accelerated bone fronts in tcf12; twist1b mutants transgenic for sp7:EGFP.

Live imaging of sp7:EGFP was performed on wild-type sibling controls (n = 11) and tcf12; twist1b mutants (n = 7) at 10 mm standard length. Dorsal views of the skull bones showed accelerated and …

https://doi.org/10.7554/eLife.37024.019
Figure 5—figure supplement 2
Additional examples of BrdU and Sp7 staining in tcf12; twist1b mutant zebrafish.

Dorsal views of 9 mm zebrafish skullcaps stained with Sp7 antibody (green) and BrdU (magenta) show an array of altered bone shapes in mutants compared to wild type. fb, frontal bone; pb, parietal …

https://doi.org/10.7554/eLife.37024.020
Figure 6 with 1 supplement
Altered bone growth dynamics in Tcf12+/-; Twist1+/- mice.

(A) Lateral views of E13.5 mouse heads show Alkaline phosphatase staining of developing frontal and parietal bones. Dotted lines indicate the bone fronts that will form the coronal suture. Compared …

https://doi.org/10.7554/eLife.37024.023
Figure 6—source data 1

Quantification of Sp7+ osteoblast number in mutant mice.

https://doi.org/10.7554/eLife.37024.026
Figure 6—source data 2

Quantification of proliferative Sp7- cells in mutant mice.

https://doi.org/10.7554/eLife.37024.027
Figure 6—source data 3

Quantification of proliferative Sp7+ osteoblasts in mutant mice.

https://doi.org/10.7554/eLife.37024.028
Figure 6—figure supplement 1
Increased calvarial bone front thickness in mutants.

(A) Sections of mouse E14.5 coronal sutures and E16.5 sagittal sutures stained for Sp7 protein (green) and DAPI (blue, nuclei) were used to assess bone thickness. Yellow lines indicate thickness …

https://doi.org/10.7554/eLife.37024.024
Figure 6—figure supplement 1—source data 1

Quantification of calvarial bone front thickness in mutant mice.

https://doi.org/10.7554/eLife.37024.025
Figure 7 with 1 supplement
Reduced osteoprogenitor pool at the mutant coronal suture.

(A, B) Sections of forming coronal and sagittal sutures of 10 mm fish and fully formed sutures of adult fish were assessed for gli1, grem1a, and prrx1a mRNA expression (white) by RNAscope in situ …

https://doi.org/10.7554/eLife.37024.029
Figure 7—figure supplement 1
Quantification of progenitor marker and Fgf pathway transcripts in tcf12; twist1b mutants.

(A) The number of gli1, grem1a, and prrx1a transcripts at the forming coronal and sagittal suture regions were quantified per region area (between the bone fronts in Figure 7A). Although the …

https://doi.org/10.7554/eLife.37024.030
Figure 7—figure supplement 1—source data 1

Quantification of gli1,grem1a, and prrx1a transcripts in coronal suture region of mutant fish.

https://doi.org/10.7554/eLife.37024.031
Figure 7—figure supplement 1—source data 2

Quantification of gli1,grem1a, and prrx1a transcripts in sagittal suture region of mutant fish.

https://doi.org/10.7554/eLife.37024.032
Figure 7—figure supplement 1—source data 3

Quantification of fgfr2 and dusp6 transcripts in coronal suture region of mutant fish.

https://doi.org/10.7554/eLife.37024.033
Tissue-autonomous bone overgrowth in Twist1 conditional mutants.

(A) Dorsal views of Alizarin-stained skulls of three-week-old mice. In the accompanying diagrams, turquoise indicates the neural-crest-derived frontal bones and gold the mesoderm-derived parietal …

https://doi.org/10.7554/eLife.37024.034
Figure 8—source data 1

Ratio of the sagittal to metopic suture length in mutant mice.

https://doi.org/10.7554/eLife.37024.035

Tables

Key resources table
Reagent type
(species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent
(D. rerio)
sp7:EGFPPMID: 20506187
RRID: ZFIN_ZDB-GENO-100402-2
Zebrafish International
Resource Center
 genetic reagent
(D. rerio)
tcf12-/-this paperallele el548; see
Supplementary file 2
Genetic reagent
(D. rerio)
twist1a-/-this paperallele el571; see
Supplementary file 2
Genetic reagent
(D. rerio)
twist1b-/-this paperallele el570; see
Supplementary file 2
Genetic reagent
(M. musculus)
Tcf12-/-PMID: 23354436Dr. Robert Maxson
(University of Southern
California)
Genetic reagent
(M. musculus)
Twist1-/-PMID: 7729687
RRID:IMSR_JAX:002221
Dr. Richard Behringer
(University of Texas, M. D.
Anderson Cancer Center)
Genetic reagent
(M. musculus)
Twist1floxPMID: 19414008
RRID:MMRRC_016842-UNC
Dr. Patrick Tam (Children's
Medical Research Institute,
The University of Sydney)
Genetic reagent
(M. musculus)
Wnt1-CrePMID: 9843687
RRID:IMSR_JAX:003829
Dr. Henry Sucov
(University of Southern
California)
Genetic reagent
(M. musculus)
Mesp1-CrePMID: 10393122Dr. Sachicko Iseki
(Tokyo Medical and
Dental University)
Antibodyrabbit anti-Osx/Sp7Santa Cruz
Biotechnology
cat.#: sc-22536-r;
RRID: AB_831618
(1:300)
Antibodyrat anti-BrdUBio-Rad Laboratoriescat.#: MCA2060 GA;
RRID: AB_10545551
(1:100–150)
Antibodygoat anti-Grem1Thermo Fisher
Scientific
cat.#: PA5-47973;
RRID: AB_2610125
(1:40)
Antibodygoat anti-Gli1R and D Systemscat.#: AF3455;
RRID: AB_2247710
(1:40)
Antibodygoat anti-rat FITCSanta Cruz
Biotechnology
cat.#: sc-2011;
RRID: AB_631753
(1:200)
Antibodygoat anti-rabbit Alexa Fluor 568Thermo Fisher
Scientific
cat.#: A-11011;
RRID: AB_143157
(1:200–500)
Antibodydonkey anti-goat Alexa Fluor 488Abcamcat.#: ab150129;
RRID: AB_2687506
(1:200)
Antibodygoat anti-rat Alexa Fluor 633Thermo Fisher
Scientific
cat.#: A21094;
RRID: AB_2535749
(1:500)
Sequence-based
reagent
RNAscope Probe - Dr-tcf12-C2Advanced Cell
Diagnostics
cat.#: 517031-C2
Sequence-based
reagent
RNAscope Probe - Dr-twist1bAdvanced Cell
Diagnostics
cat.#: 413121
Sequence-based
reagent
RNAscope Probe - Dr-gli1-C3Advanced Cell
Diagnostics
(not yet in catalog)
Sequence-based
reagent
RNAscope Probe - Dr-grem1aAdvanced Cell
Diagnostics
cat.#: 535291
Sequence-based
reagent
RNAscope Probe - Dr-prrx1aAdvanced Cell
Diagnostics
cat.#: 535321
Sequence-based
reagent
RNAscope Probe - Dr-fgfr2Advanced Cell
Diagnostics
cat.#: 420961
Sequence-based
reagent
RNAscope Probe - Dr-dusp6-C3Advanced Cell
Diagnostics
cat.#: 515021-C3
Commercial assay
or kit
RNAscope 2.5 HD Assay – REDAdvanced Cell
Diagnostics
cat.#: 322350
Commercial assay
or kit
RNAscope Multiplex Fluorescent Kit v2Advanced Cell
Diagnostics
cat.#: 323110
Chemical compound,
drug
Alizarin Red SAmrescocat.#: 9436–25Glive staining: 1 mg / 30 mL
Chemical compound,
drug
CalceinThermo Fisher
Scientific
cat.#: C481live staining: 1 mg / 10 mL
Chemical compound,
drug
Alcian BlueAnatech LTDcat.#: 862

Additional files

Supplementary file 1

Summary of phenotypes observed in combinatorial zebrafish mutants

https://doi.org/10.7554/eLife.37024.036
Supplementary file 2

TALEN targeting and mutant genotyping

https://doi.org/10.7554/eLife.37024.037
Transparent reporting form
https://doi.org/10.7554/eLife.37024.038

Download links