(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 …
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 …
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 …
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 …
(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. …
Cartilage size in twist1a;twist1b mutants.
Severity of cartilage defects in combinatorial Twist1 and Tcf12 mutants.
Adult viability of combinatorial Twist1 and Tcf12 mutants.
(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 …
(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 …
Quantification of skull bone area in mutants by 10.25 mm.
Directionality of bone growth in mutants.
Quantification of growth in coronal area.
Quantification of growth in metopic area.
Quantification of growth in sagittal area.
(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 …
Quantification of proliferative sp7+ osteoblasts in mutant zebrafish.
Quantification of proliferative sp7- cells in mutant zebrafish.
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 …
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 …
(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 …
Quantification of Sp7+ osteoblast number in mutant mice.
Quantification of proliferative Sp7- cells in mutant mice.
Quantification of proliferative Sp7+ osteoblasts in mutant mice.
(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 …
Quantification of calvarial bone front thickness in mutant mice.
(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 …
(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 …
Quantification of gli1,grem1a, and prrx1a transcripts in coronal suture region of mutant fish.
Quantification of gli1,grem1a, and prrx1a transcripts in sagittal suture region of mutant fish.
Quantification of fgfr2 and dusp6 transcripts in coronal suture region of mutant fish.
(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 …
Ratio of the sagittal to metopic suture length in mutant mice.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Genetic reagent (D. rerio) | sp7:EGFP | PMID: 20506187 | RRID: ZFIN_ZDB-GENO-100402-2 | Zebrafish International Resource Center |
genetic reagent (D. rerio) | tcf12-/- | this paper | allele el548; see Supplementary file 2 | |
Genetic reagent (D. rerio) | twist1a-/- | this paper | allele el571; see Supplementary file 2 | |
Genetic reagent (D. rerio) | twist1b-/- | this paper | allele el570; see Supplementary file 2 | |
Genetic reagent (M. musculus) | Tcf12-/- | PMID: 23354436 | Dr. 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) | Twist1flox | PMID: 19414008 | RRID:MMRRC_016842-UNC | Dr. Patrick Tam (Children's Medical Research Institute, The University of Sydney) |
Genetic reagent (M. musculus) | Wnt1-Cre | PMID: 9843687 | RRID:IMSR_JAX:003829 | Dr. Henry Sucov (University of Southern California) |
Genetic reagent (M. musculus) | Mesp1-Cre | PMID: 10393122 | Dr. Sachicko Iseki (Tokyo Medical and Dental University) | |
Antibody | rabbit anti-Osx/Sp7 | Santa Cruz Biotechnology | cat.#: sc-22536-r; RRID: AB_831618 | (1:300) |
Antibody | rat anti-BrdU | Bio-Rad Laboratories | cat.#: MCA2060 GA; RRID: AB_10545551 | (1:100–150) |
Antibody | goat anti-Grem1 | Thermo Fisher Scientific | cat.#: PA5-47973; RRID: AB_2610125 | (1:40) |
Antibody | goat anti-Gli1 | R and D Systems | cat.#: AF3455; RRID: AB_2247710 | (1:40) |
Antibody | goat anti-rat FITC | Santa Cruz Biotechnology | cat.#: sc-2011; RRID: AB_631753 | (1:200) |
Antibody | goat anti-rabbit Alexa Fluor 568 | Thermo Fisher Scientific | cat.#: A-11011; RRID: AB_143157 | (1:200–500) |
Antibody | donkey anti-goat Alexa Fluor 488 | Abcam | cat.#: ab150129; RRID: AB_2687506 | (1:200) |
Antibody | goat anti-rat Alexa Fluor 633 | Thermo Fisher Scientific | cat.#: A21094; RRID: AB_2535749 | (1:500) |
Sequence-based reagent | RNAscope Probe - Dr-tcf12-C2 | Advanced Cell Diagnostics | cat.#: 517031-C2 | |
Sequence-based reagent | RNAscope Probe - Dr-twist1b | Advanced Cell Diagnostics | cat.#: 413121 | |
Sequence-based reagent | RNAscope Probe - Dr-gli1-C3 | Advanced Cell Diagnostics | (not yet in catalog) | |
Sequence-based reagent | RNAscope Probe - Dr-grem1a | Advanced Cell Diagnostics | cat.#: 535291 | |
Sequence-based reagent | RNAscope Probe - Dr-prrx1a | Advanced Cell Diagnostics | cat.#: 535321 | |
Sequence-based reagent | RNAscope Probe - Dr-fgfr2 | Advanced Cell Diagnostics | cat.#: 420961 | |
Sequence-based reagent | RNAscope Probe - Dr-dusp6-C3 | Advanced Cell Diagnostics | cat.#: 515021-C3 | |
Commercial assay or kit | RNAscope 2.5 HD Assay – RED | Advanced Cell Diagnostics | cat.#: 322350 | |
Commercial assay or kit | RNAscope Multiplex Fluorescent Kit v2 | Advanced Cell Diagnostics | cat.#: 323110 | |
Chemical compound, drug | Alizarin Red S | Amresco | cat.#: 9436–25G | live staining: 1 mg / 30 mL |
Chemical compound, drug | Calcein | Thermo Fisher Scientific | cat.#: C481 | live staining: 1 mg / 10 mL |
Chemical compound, drug | Alcian Blue | Anatech LTD | cat.#: 862 |
Summary of phenotypes observed in combinatorial zebrafish mutants
TALEN targeting and mutant genotyping