Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jun;56(6-7):e23220.
doi: 10.1002/dvg.23220.

Rapamycin rescues BMP mediated midline craniosynostosis phenotype through reduction of mTOR signaling in a mouse model

Kaitrin Kramer  1 , Jingwen Yang  1 , W Benton Swanson, Satoru Hayano  1   2 , Masako Toda  1 , Haichun Pan  1 , Jin Koo Kim  1   3 , Paul H Krebsbach  1   3 , Yuji Mishina  1
Affiliations

Rapamycin rescues BMP mediated midline craniosynostosis phenotype through reduction of mTOR signaling in a mouse model

Kaitrin Kramer et al. Genesis. 2018 Jun.

Erratum in

  • Erratum.
    [No authors listed] [No authors listed] Genesis. 2022 Jul;60(6-7):e23489. doi: 10.1002/dvg.23489. Epub 2022 Jun 20. Genesis. 2022. PMID: 35723232 No abstract available.

Abstract

Craniosynostosis is defined as congenital premature fusion of one or more cranial sutures. While the genetic basis for about 30% of cases is known, the causative genes for the diverse presentations of the remainder of cases are unknown. The recently discovered cranial suture stem cell population affords an opportunity to identify early signaling pathways that contribute to craniosynostosis. We previously demonstrated that enhanced BMP signaling in neural crest cells (caA3 mutants) leads to premature cranial suture fusion resulting in midline craniosynostosis. Since enhanced mTOR signaling in neural crest cells leads to craniofacial bone lesions, we investigated the extent to which mTOR signaling is involved in the pathogenesis of BMP-mediated craniosynostosis by affecting the suture stem cell population. Our results demonstrate a loss of suture stem cells in the caA3 mutant mice by the newborn stage. We have found increased activation of mTOR signaling in caA3 mutant mice during embryonic stages, but not at the newborn stage. Our study demonstrated that inhibition of mTOR signaling via rapamycin in a time specific manner partially rescued the loss of the suture stem cell population. This study provides insight into how enhanced BMP signaling regulates suture stem cells via mTOR activation.

Keywords: BMP Smad signaling; craniosynostosis; mTOR; neural crest cells; suture.

PubMed Disclaimer

Conflict of interest statement

All authors state that they have no conflicts of interest.

Figures

Figure 1
Figure 1. Reduction of suture stem cell population in caA3 mutant mice (caA3 mut)
A. Whole heads of caA3 mut mice carrying Gli1-lacZ were stained with X-gal at newborn stage (a, d), postnatal (PN) day 6 (b, e) and day 20 (c, f). Approximate positions of sutures are shown in (a). af, anterior frontal; cr, coronal; fm, frontomaxillary; fms, frontal-maxillary-sphenoid; lm, lambdoid; n, nasal; pf, posterior frontal. Arrowheads in d, e, loss of Gli1 expression in the nasal suture; arrows in d, e, f, loss of Gli1 expression in the frontal suture. B. Histological observations at different anterior-posterior levels. Arrows in (a) and (d) indicate the nasal suture. Areas pointed by arrows in (b) and (e) are enlarged at (c) and (f). Areas pointed by arrows in (g) and (j) are enlarged at (h) and (k). Boxes in (g) and (j) are enlarged at (i) and (l). fr, frontal bone; mx, maxilla; ns, nasal bone; sp; sphenoid. Bar = 100 μm.
Figure 2
Figure 2. mTOR signaling is increased in the caA3 mut embryos
A. Schematic representation of mTOR signaling pathway. B. Protein lysate prepared from the head region of control and caA3 mut embryos (E10.5) were subjected for Western blot analyses to quantify levels of mTOR signaling components. C. Levels of phosphorylation of each signaling component normalized by total proteins were quantified. Fold increases of caA3 mutants compared with controls are shown. n=3. *, p<0.05; **, p<0.01. D. mTOR signaling levels were examined using a phospho-S6 antibody on frontal sections of cranial regions of E10.5 embryos. Nuclear DNA was stained with DAPI (blue). LNP, lateral nasal process, MNP, medial nasal process, NT, neural tube, *, presumptive choroid plexus. Approximate positions of each section are also shown (a, b, nasal level, c, d, before eye level). Bar = 100 μm.
Figure 3
Figure 3. mTOR signaling is transiently increased during embryogenesis
mTOR signaling levels were examined using a phospho-S6 antibody on frontal sections of cranial regions from E13.5 (A, eye level), E17.5 (B, anterior frontal suture level) and newborn (NB) (C, D, nasal and anterior suture levels, respectively) mice. Nuclear DNA was stained with DAPI (blue). White arrows in A indicate different signaling levels at the surface of embryos. White boxes in B indicate presumptive suture area negative for mTOR signaling in control embryos. White boxes in C and D indicate suture areas where control mice show more intense immunosignals. Quantification of mean fluorescence of the suture area in caA3 mutant and control images for E17.5, NB nasal and NB Anterior Frontal shown (E) *, p<0.05; n>3 per group. Bar = 500 μm.
Figure 4
Figure 4. Rapamycin treatment reduces facial abnormalities caused by increased BMP signaling
A. Three injection regimens for rapamycin treatment. Tx 1, 1 mg/kg daily injection from E14.5 to E18.5. Tx 2, 1 mg/kg daily injection from E8.5 to E18.5. Tx 3, 5 mg/kg at E8.5 only. B. Immunohistochemistry for pS6 at newborn stage showed reduction of mTOR signaling by rapamycin treatment in caA3 mutants (Tx 1). C. Lateral view of newborn stage pups. Typical images of vehicle treated (Vehicle Tx) and rapamycin treated (Tx 3, Rapamycin Tx) pups are shown. D. Measurement of the naso-frontal angle. Rapamycin treated caA3 mut embryos using Tx 1 did not show significant improvement while treated using Tx 2 and Tx 3 showed significant improvement on this measurement. n> 6 for each group. n.s., no significance; *, p<0.05; **p<0.01; ***, p<0.001. E. Measurement of the nasal and frontal lengths. Actual lengths are normalized by ear widths of each mice. Rapamycin treated caA3 mutant embryos using Tx 1 did not show significant improvement while treated using Tx 2 and Tx 3 showed tendency of improvement on the relative nasal length. No significant changes were found in the relative frontal length between genotypes and treatments. n> 6 for each group. n.s., no significance; *, p<0.05; **p<0.01; ***, p<0.001.
Figure 5
Figure 5. Rapamycin treatment reduces distance between skull bones
A. control and caA3 mut newborn skulls were stained with X-gal after vehicle treatment or rapamycin treatment (Tx 3, 5 mg/kg at E8.5). B. caA3 mut newborn skulls treated with rapamycin (Tx 1, 1mg/kg daily injection from E14.5 or Tx 2, 1 mg/kg daily injection from E8.5) C. Distance between the frontal bones (a) and between the parietal bones (b) were measured. n> 3 for each group. #, p=0.1, *, p<0.05; **p<0.01; ***, p<0.001. D. Lengths of the nasal (c) and the frontal (d) bones and the distance between eyes were measured. n> 3 for each group. *, p<0.05; **p<0.01; ***, p<0.001.
Figure 6
Figure 6. Suppression of cell death in the suture mesenchyme by rapamycin treatment
Frontal sections of lacZ (blue) stained heads from new born stage mice were made and levels of cell death were measured by TUNEL (orange). The nasal suture (upper panels) and the anterior frontal suture (lower panels) from control and caA3 mutant mice received different treatments are shown. cont, caBmpr1a(+);P0-Cre(−), caA3 mut, caBmpr1a(+);P0-Cre(+). Bar = 100 μm.
Figure 7
Figure 7. An increase of mTOR signaling did not change morphologies
A. Lateral view of newborn control (caBmpr1a(+);P0-Cre(−)) and caA3 mut (caBmpr1a(+);P0-Cre(+)) mice with or without heterozygous null mutation of Tsc1 (Tsc1+/− or Tsc1+/+). B. Top view of skull after X-gal staining. C. Morphometric measurements. The naso-frontal angle (described in Fig. 4), distance between the frontal bones, ratios of distances between the frontal bones to those between the parietal bones, lengths of the nasal and the frontal bones and the distance between eyes (described in Fig. 5) were measured. n> 11 for each group. *, p<0.05; **p<0.01; n.s., no significance. D. Western blot analyses of E10.5 embryonic heads for mTOR signaling activity. Each band intensity was normalized by GAPDH. Typical examples of each genotype were shown.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Agarwal S, Loder SJ, Brownley C, Eboda O, Peterson JR, Hayano S, … Levi B. BMP signaling mediated by constitutively active Activin type 1 receptor (ACVR1) results in ectopic bone formation localized to distal extremity joints. Dev Biol. 2015;400(2):202–209. doi: 10.1016/j.ydbio.2015.02.011. - DOI - PMC - PubMed
    1. Arriola Apelo SI, Neuman JC, Baar EL, Syed FA, Cummings NE, Brar HK, … Lamming DW. Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system. Aging Cell. 2016;15(1):28–38. doi: 10.1111/acel.12405. - DOI - PMC - PubMed
    1. Badve CA, KMM, Iyer RS, Ishak GE, Khanna PC. Craniosynostosis: imaging review and primer on computed tomography. Pediatr Radiol. 2013;43(6):728–742. doi: 10.1007/s00247-013-2673-6. quiz 725–727. - DOI - PubMed
    1. Bai CB, Auerbach W, Lee JS, Stephen D, Joyner AL. Gli2, but not Gli1, is required for initial Shh signaling and ectopic activation of the Shh pathway. Development. 2002;129(20):4753–4761. - PubMed
    1. Briscoe J, Pierani A, Jessell TM, Ericson J. A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell. 2000;101(4):435–445. - PubMed

Publication types