Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Muscle connective tissue controls development of the diaphragm and is a source of congenital diaphragmatic hernias

Nature Genetics volume 47, pages 496–504 (2015)Cite this article

Subjects

Abstract

The diaphragm is an essential mammalian skeletal muscle, and defects in diaphragm development are the cause of congenital diaphragmatic hernias (CDHs), a common and often lethal birth defect. The diaphragm is derived from multiple embryonic sources, but how these give rise to the diaphragm is unknown, and, despite the identification of many CDH-associated genes, the etiology of CDH is incompletely understood. Using mouse genetics, we show that the pleuroperitoneal folds (PPFs), which are transient embryonic structures, are the source of the diaphragm's muscle connective tissue and regulate muscle development, and we show that the striking migration of PPF cells controls diaphragm morphogenesis. Furthermore, Gata4 mosaic mutations in PPF-derived muscle connective tissue fibroblasts result in the development of localized amuscular regions that are biomechanically weaker and more compliant, leading to CDH. Thus, the PPFs and muscle connective tissue are critical for diaphragm development, and mutations in PPF-derived fibroblasts are a source of CDH.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: PPFs contain Gata4+ muscle connective tissue fibroblasts that migrate independently and in advance of myogenic cells.
Figure 2: Deletion of Gata4 in the PPFs produces localized amuscular regions that are weaker than juxtaposed muscular regions and results in CDH.
Figure 3: In CDH, physical impedance by herniated tissue causes lung hypoplasia.
Figure 4: CDH results from early defects in the localization of muscle progenitors.
Figure 5: Hgf is strongly expressed in PPF cells and downregulated in Gata4-null fibroblasts.
Figure 6: Early defects in proliferation, apoptosis and localization of muscle progenitors lead to CDH.
Figure 7: Model of CDH development.

Similar content being viewed by others

References

  1. Perry, S.F., Similowski, T., Klein, W. & Codd, J.R. The evolutionary origin of the mammalian diaphragm. Respir. Physiol. Neurobiol. 171, 1–16 (2010).

    Article  Google Scholar 

  2. Campbell, E.J.M., Agostoni, E. & Newsom Davis, J. The Respiratory Muscles: Mechanics and Neural Control (Lloyd-Luke, 1970).

  3. Merrell, A.J. & Kardon, G. Development of the diaphragm—a skeletal muscle essential for mammalian respiration. FEBS J. 280, 4026–4035 (2013).

    Article  CAS  Google Scholar 

  4. Raval, M.V., Wang, X., Reynolds, M. & Fischer, A.C. Costs of congenital diaphragmatic hernia repair in the United States—extracorporeal membrane oxygenation foots the bill. J. Pediatr. Surg. 46, 617–624 (2011).

    Article  Google Scholar 

  5. Torfs, C.P., Curry, C.J., Bateson, T.F. & Honore, L.H. A population-based study of congenital diaphragmatic hernia. Teratology 46, 555–565 (1992).

    Article  CAS  Google Scholar 

  6. Pober, B.R. Overview of epidemiology, genetics, birth defects, and chromosome abnormalities associated with CDH. Am. J. Med. Genet. C. Semin. Med. Genet. 145C, 158–171 (2007).

    Article  CAS  Google Scholar 

  7. Pober, B.R. et al. Infants with Bochdalek diaphragmatic hernia: sibling precurrence and monozygotic twin discordance in a hospital-based malformation surveillance program. Am. J. Med. Genet. A. 138A, 81–88 (2005).

    Article  Google Scholar 

  8. Holder, A.M. et al. Genetic factors in congenital diaphragmatic hernia. Am. J. Hum. Genet. 80, 825–845 (2007).

    Article  CAS  Google Scholar 

  9. Veenma, D.C., de Klein, A. & Tibboel, D. Developmental and genetic aspects of congenital diaphragmatic hernia. Pediatr. Pulmonol. 47, 534–545 (2012).

    Article  CAS  Google Scholar 

  10. Russell, M.K. et al. Congenital diaphragmatic hernia candidate genes derived from embryonic transcriptomes. Proc. Natl. Acad. Sci. USA 109, 2978–2983 (2012).

    Article  CAS  Google Scholar 

  11. Arrington, C.B. et al. A family-based paradigm to identify candidate chromosomal regions for isolated congenital diaphragmatic hernia. Am. J. Med. Genet. A. 158A, 3137–3147 (2012).

    Article  Google Scholar 

  12. Longoni, M. et al. Congenital diaphragmatic hernia interval on chromosome 8p23.1 characterized by genetics and protein interaction networks. Am. J. Med. Genet. A. 158A, 3148–3158 (2012).

    Article  Google Scholar 

  13. Yu, L. et al. Variants in GATA4 are a rare cause of familial and sporadic congenital diaphragmatic hernia. Hum. Genet. 132, 285–292 (2013).

    Article  CAS  Google Scholar 

  14. Kuo, C.T. et al. GATA4 transcription factor is required for ventral morphogenesis and heart tube formation. Genes Dev. 11, 1048–1060 (1997).

    Article  CAS  Google Scholar 

  15. Molkentin, J.D., Lin, Q., Duncan, S.A. & Olson, E.N. Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis. Genes Dev. 11, 1061–1072 (1997).

    Article  CAS  Google Scholar 

  16. Jay, P.Y. et al. Impaired mesenchymal cell function in Gata4 mutant mice leads to diaphragmatic hernias and primary lung defects. Dev. Biol. 301, 602–614 (2007).

    Article  CAS  Google Scholar 

  17. Mendelsohn, C. et al. Function of the retinoic acid receptors (RARs) during development (II). Multiple abnormalities at various stages of organogenesis in RAR double mutants. Development 120, 2749–2771 (1994).

    CAS  PubMed  Google Scholar 

  18. You, L.R. et al. Mouse lacking COUP-TFII as an animal model of Bochdalek-type congenital diaphragmatic hernia. Proc. Natl. Acad. Sci. USA 102, 16351–16356 (2005).

    Article  CAS  Google Scholar 

  19. Ackerman, K.G. et al. Fog2 is required for normal diaphragm and lung development in mice and humans. PLoS Genet. 1, 58–65 (2005).

    Article  CAS  Google Scholar 

  20. Coles, G.L. & Ackerman, K.G. Kif7 is required for the patterning and differentiation of the diaphragm in a model of syndromic congenital diaphragmatic hernia. Proc. Natl. Acad. Sci. USA 110, E1898–E1905 (2013).

    Article  CAS  Google Scholar 

  21. Allan, D.W. & Greer, J.J. Embryogenesis of the phrenic nerve and diaphragm in the fetal rat. J. Comp. Neurol. 382, 459–468 (1997).

    Article  CAS  Google Scholar 

  22. Babiuk, R.P., Zhang, W., Clugston, R., Allan, D.W. & Greer, J.J. Embryological origins and development of the rat diaphragm. J. Comp. Neurol. 455, 477–487 (2003).

    Article  Google Scholar 

  23. Dietrich, S. et al. The role of SF/HGF and c-Met in the development of skeletal muscle. Development 126, 1621–1629 (1999).

    CAS  PubMed  Google Scholar 

  24. Greer, J.J. et al. Structure of the primordial diaphragm and defects associated with nitrofen-induced CDH. J. Appl. Physiol. 89, 2123–2129 (2000).

    Article  CAS  Google Scholar 

  25. Ackerman, K.G. & Greer, J.J. Development of the diaphragm and genetic mouse models of diaphragmatic defects. Am. J. Med. Genet. C. Semin. Med. Genet. 145C, 109–116 (2007).

    Article  CAS  Google Scholar 

  26. Logan, M. et al. Expression of Cre recombinase in the developing mouse limb bud driven by a Prx1 enhancer. Genesis 33, 77–80 (2002).

    Article  CAS  Google Scholar 

  27. Soriano, P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70–71 (1999).

    Article  CAS  Google Scholar 

  28. Kardon, G., Harfe, B.D. & Tabin, C.J.A. Tcf4-positive mesodermal population provides a prepattern for vertebrate limb muscle patterning. Dev. Cell 5, 937–944 (2003).

    Article  CAS  Google Scholar 

  29. Mathew, S.J. et al. Connective tissue fibroblasts and Tcf4 regulate myogenesis. Development 138, 371–384 (2011).

    Article  CAS  Google Scholar 

  30. Clugston, R.D., Zhang, W. & Greer, J.J. Gene expression in the developing diaphragm: significance for congenital diaphragmatic hernia. Am. J. Physiol. Lung Cell. Mol. Physiol. 294, L665–L675 (2008).

    Article  CAS  Google Scholar 

  31. Engleka, K.A. et al. Insertion of Cre into the Pax3 locus creates a new allele of Splotch and identifies unexpected Pax3 derivatives. Dev. Biol. 280, 396–406 (2005).

    Article  CAS  Google Scholar 

  32. Vogan, K.J., Epstein, D.J., Trasler, D.G. & Gros, P. The splotch-delayed (Spd) mouse mutant carries a point mutation within the paired box of the Pax-3 gene. Genomics 17, 364–369 (1993).

    Article  CAS  Google Scholar 

  33. Watt, A.J., Battle, M.A., Li, J. & Duncan, S.A. GATA4 is essential for formation of the proepicardium and regulates cardiogenesis. Proc. Natl. Acad. Sci. USA 101, 12573–12578 (2004).

    Article  CAS  Google Scholar 

  34. Ackerman, K.G. et al. Congenital diaphragmatic defects: proposal for a new classification based on observations in 234 patients. Pediatr. Dev. Pathol. 15, 265–274 (2012).

    Article  Google Scholar 

  35. Clugston, R.D. & Greer, J.J. Diaphragm development and congenital diaphragmatic hernia. Semin. Pediatr. Surg. 16, 94–100 (2007).

    Article  Google Scholar 

  36. Maas, S.A., Ellis, B.J., Ateshian, G.A. & Weiss, J.A. FEBio: finite elements for biomechanics. J. Biomech. Eng. 134, 011005 (2012).

    Article  Google Scholar 

  37. Strumpf, R.K., Humphrey, J.D. & Yin, F.C. Biaxial mechanical properties of passive and tetanized canine diaphragm. Am. J. Physiol. 265, H469–H475 (1993).

    CAS  PubMed  Google Scholar 

  38. Keijzer, R., Liu, J., Deimling, J., Tibboel, D. & Post, M. Dual-hit hypothesis explains pulmonary hypoplasia in the nitrofen model of congenital diaphragmatic hernia. Am. J. Pathol. 156, 1299–1306 (2000).

    Article  CAS  Google Scholar 

  39. Ackerman, K.G. et al. Gata4 is necessary for normal pulmonary lobar development. Am. J. Respir. Cell Mol. Biol. 36, 391–397 (2007).

    Article  CAS  Google Scholar 

  40. Bladt, F., Riethmacher, D., Isenmann, S., Aguzzi, A. & Birchmeier, C. Essential role for the c-Met receptor in the migration of myogenic precursor cells into the limb bud. Nature 376, 768–771 (1995).

    Article  CAS  Google Scholar 

  41. Maina, F. et al. Uncoupling of Grb2 from the Met receptor in vivo reveals complex roles in muscle development. Cell 87, 531–542 (1996).

    Article  CAS  Google Scholar 

  42. Nakamura, K., Hongo, A., Kodama, J. & Hiramatsu, Y. The role of hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2 in endometrial cancer. Int. J. Cancer 128, 2613–2624 (2011).

    Article  CAS  Google Scholar 

  43. Rojas, A. et al. GATA4 is a direct transcriptional activator of cyclin D2 and Cdk4 and is required for cardiomyocyte proliferation in anterior heart field–derived myocardium. Mol. Cell. Biol. 28, 5420–5431 (2008).

    Article  CAS  Google Scholar 

  44. Yamak, A. et al. Cyclin D2 rescues size and function of GATA4 haplo-insufficient hearts. Am. J. Physiol. Heart Circ. Physiol. 303, H1057–H1066 (2012).

    Article  CAS  Google Scholar 

  45. Domyan, E.T. et al. Roundabout receptors are critical for foregut separation from the body wall. Dev. Cell 24, 52–63 (2013).

    Article  CAS  Google Scholar 

  46. Zhang, B. et al. Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia. J. Clin. Invest. 124, 209–221 (2014).

    Article  CAS  Google Scholar 

  47. Veenma, D. et al. Copy number detection in discordant monozygotic twins of congenital diaphragmatic hernia (CDH) and esophageal atresia (EA) cohorts. Eur. J. Hum. Genet. 20, 298–304 (2012).

    Article  CAS  Google Scholar 

  48. Wat, M.J. et al. Chromosome 8p23.1 deletions as a cause of complex congenital heart defects and diaphragmatic hernia. Am. J. Med. Genet. A. 149A, 1661–1677 (2009).

    Article  CAS  Google Scholar 

  49. Kantarci, S. et al. Characterization of the chromosome 1q41q42.12 region, and the candidate gene DISP1, in patients with CDH. Am. J. Med. Genet. A. 152A, 2493–2504 (2010).

    Article  CAS  Google Scholar 

  50. Veenma, D. et al. Comparable low-level mosaicism in affected and non affected tissue of a complex CDH patient. PLoS ONE 5, e15348 (2010).

    Article  CAS  Google Scholar 

  51. Pu, W.T., Ishiwata, T., Juraszek, A.L., Ma, Q. & Izumo, S. GATA4 is a dosage-sensitive regulator of cardiac morphogenesis. Dev. Biol. 275, 235–244 (2004).

    Article  CAS  Google Scholar 

  52. Wat, M.J. et al. Mouse model reveals the role of SOX7 in the development of congenital diaphragmatic hernia associated with recurrent deletions of 8p23.1. Hum. Mol. Genet. 21, 4115–4125 (2012).

    Article  CAS  Google Scholar 

  53. Bosch, N. et al. Nucleotide, cytogenetic and expression impact of the human chromosome 8p23.1 inversion polymorphism. PLoS ONE 4, e8269 (2009).

    Article  Google Scholar 

  54. Giglio, S. et al. Olfactory receptor-gene clusters, genomic-inversion polymorphisms, and common chromosome rearrangements. Am. J. Hum. Genet. 68, 874–883 (2001).

    Article  CAS  Google Scholar 

  55. Reamon-Buettner, S.M. & Borlak, J. GATA4 zinc finger mutations as a molecular rationale for septation defects of the human heart. J. Med. Genet. 42, e32 (2005).

    Article  CAS  Google Scholar 

  56. Reamon-Buettner, S.M., Cho, S.H. & Borlak, J. Mutations in the 3′-untranslated region of GATA4 as molecular hotspots for congenital heart disease (CHD). BMC Med. Genet. 8, 38 (2007).

    Article  Google Scholar 

  57. Biesecker, L.G. & Spinner, N.B. A genomic view of mosaicism and human disease. Nat. Rev. Genet. 14, 307–320 (2013).

    Article  CAS  Google Scholar 

  58. Tang, S.H., Silva, F.J., Tsark, W.M. & Mann, J.R.A. Cre/loxP-deleter transgenic line in mouse strain 129S1/SvImJ. Genesis 32, 199–202 (2002).

    Article  CAS  Google Scholar 

  59. Muzumdar, M.D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007).

    Article  CAS  Google Scholar 

  60. Wan, Y., Otsuna, H., Chien, C.B. & Hansen, C. An interactive visualization tool for multi-channel confocal microscopy data in neurobiology research. IEEE Trans. Vis. Comput. Graph. 15, 1489–1496 (2009).

    Article  Google Scholar 

  61. Veronda, D.R. & Westmann, R.A. Mechanical characterization of skin-finite deformations. J. Biomech. 3, 111–124 (1970).

    Article  CAS  Google Scholar 

  62. Mooney, M. A theory of large elastic deformation. J. Appl. Phys. 11, 582–592 (1940).

    Article  Google Scholar 

Download references

Acknowledgements

We thank C. Rodesch at the University of Utah Imaging Core for help with microscopy, S. Merchant for help with microCT imaging, Y. Wan and C. Hansen for Fluorender analysis, M. Hockin and M.R. Capecchi for Cre protein, and M. Colasanto, N. Elde, L.B. Jorde, A. Keefe, A. Letsou, L.C. Murtaugh and C.J. Tabin for critical comments on the manuscript. A.J.M. was supported by a University of Utah Graduate Fellowship, FEBio analysis is supported by US National Institutes of Health (NIH) grant R01GM083925 to J.A.W. and G.A. Ateshian, and Fluorender analysis is supported by US NIH grant R01GM098151 to C. Hansen. This research was supported by US NIH grant R01HD053728 and March of Dimes grant FY12-405 to G.K.

Author information

Author notes

  1. Benjamin J Ellis and Zachary D Fox: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Human Genetics, University of Utah, Salt Lake City, Utah, USA

    Allyson J Merrell, Zachary D Fox, Jennifer A Lawson & Gabrielle Kardon

  2. Department of Bioengineering, University of Utah, Salt Lake City, Utah, USA

    Benjamin J Ellis & Jeffrey A Weiss

  3. Scientific Computing and Imaging Institute, University of Utah, Salt Lake City, Utah, USA

    Benjamin J Ellis & Jeffrey A Weiss

Authors
  1. Allyson J Merrell
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Benjamin J Ellis
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zachary D Fox
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jennifer A Lawson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jeffrey A Weiss
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gabrielle Kardon
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.J.M. conducted experiments, analyzed data and wrote the manuscript. Z.D.F. conducted two-photon experiments. B.J.E. and J.A.W. contributed finite element model analysis. J.A.L. managed the mouse colony. G.K. conducted experiments, analyzed data and wrote the manuscript.

Corresponding author

Correspondence to Gabrielle Kardon.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 PPF cells migrate dynamically over the liver and septum transversum.

(a) Trajectories of individual GFP+ PPF cells labeled in E12.5 Prx1-creTg/+; RosamTmG/+ diaphragm explanted, cultured and imaged via two-photon microscopy for 4.6 h. Each gray arrow shows the absolute movement of each cell during 4.6 h, and each black arrow shows the movement of each cell normalized by the movement of the body wall. Arrows on the right were normalized by the average movement of the right body wall (arrow within the red circle), and arrows on the left were normalized by the average movement of the left body wall (arrow within the green circle). (b) Movement of individual cells during 4.6 h (boxed region in a). The line of dots shows the movement of each cell during the nine 30.7-min intervals. (a,b) Movement of the cells shown in Supplementary Video 2. The color-coded trajectories correspond to the color-coded cells in Supplementary Video 2. Scale bars, 500 μm (a), 125 μm (b).

Supplementary Figure 2 Genetic labeling and ablation with the Prx1-cre transgene.

(a) The Prx1-cre transgene labels some cells in the lungs (n > 3/3). (b,c) Lung alveoli appear largely unaffected in mutant Prx1-creTg/+; Gata4Δ/fl mice. (d–i) The Prx1-cre transgene causes incomplete deletion of Gata4 in the PPFs at E12.5 (n = 4/4). Left insets show incomplete Prx1-cre–mediated recombination of Rosa26LacZ and Gata4fl, and right insets show more complete Prx1-cre–mediated recombination of Rosa26LacZ and Gata4fl. (a) Whole-mount β-galactosidase staining. (b–g) Section immunofluorescence. Scale bars, 1 mm (a), 50 μm (b,c), 100 μm (d–i), 20 μm (insets in d–i).

Supplementary Figure 3 Cell cycle proteins are downregulated in PPF cells with deletion of Gata4.

(a–l) Cdk4 (a–f) and cyclin D2 (g–l) are downregulated in Gata4-null PPF cells. E12.5 section immunofluorescence. Scale bars, 100 μm (a–l), 20 μm (insets in a–l).

Supplementary Figure 4 Gata4-null fibroblasts inhibit the proliferation of myogenic cells.

(a) When cultured, Gata4-null, as compared to wild-type, PPF fibroblasts proliferate less (n = 3 technical replicates). (b,c) When cultured for 48 h with Gata4-null PPF fibroblasts, as compared with Gata4-heterozygous fibroblasts, there are decreased cell numbers (b; n = 5 biological replicates, each with 3 technical replicates) and proliferation (c; n = 3 technical replicates) of Pax7+MyoD+ diaphragmatic myogenic cells. Micrographs show immunofluorescence labeling of fibroblasts (a) or myogenic cells (b,c). Scale bar, 50 μm. For charts, bars show mean values ± 1 s.e.m. P-values are from two-tailed Student’s t tests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Table 1. (PDF 1441 kb)

PPF cells actively migrate during diaphragm morphogenesis.

5.5-h time-lapse. GFP+ PPF cells at the leading edge migrate ventrally and medially (upper left to lower right in the video) across the surface of the liver in an explant of E12.5 Prx1-creTg/+; RosamTmG/+ diaphragm (with attached ribs and underlying liver). The location corresponding to the video is indicated by an asterisk in Figure 1c. The interval between frames is 5.6 min. The two red lines show the trajectory of the midpoint of the two labeled cells. Note that in frame 17 the entire diaphragm shifted down and at the end of the movie the diaphragm gradually sinks out of the field of view. Scale bar, 50 μm. (MOV 10814 kb)

PPF cells actively migrate across the liver surface during diaphragm morphogenesis.

4.6-h time-lapse of GFP+ PPF cells migrating ventrally across the surface of the liver in an explant of E12.5 Prx1-creTg/+; RosamTmG/+ diaphragm (with attached ribs and underlying liver). Ten individual fibroblasts are color-coded and tracked. The trajectories of selected cells are shown in Supplementary Figure 1. Scale bar, 500 μm. (MOV 5065 kb)

FEM modeling demonstrates that a hernia develops when the amuscular region is more compliant than the surrounding muscular region.

Top view. (MOV 231 kb)

FEM modeling demonstrates that a hernia develops when an amuscular region is more compliant than the surrounding muscular region.

Cross-sectional view. (MOV 116 kb)

Normal morphology of embryonic mouse lungs as shown by microCT.

Lateral view of the E18.5 lungs of control Prx1-creTg/+; Gata4fl/+ mice, with right lungs shown in blue. (MOV 7236 kb)

Lungs are malformed in regions associated with hernias as shown by microCT.

Lateral E18.5 lungs of mutant Prx1-creTg/+; Gata4fl/+ mice, with right lungs shown in blue. The divot in right lungs corresponds to the herniated region. (MOV 6200 kb)

Costal muscle progenitors normally develop within the pleuroperitoneal folds.

At E12.5, myogenic progenitors normally develop intermingled with and surrounded by PPF cells, and low levels of apoptotic cells are present in control Prx1-creTg/+; Gata4fl/+; RosamTmG/+ diaphragms. Three-dimensional view of whole-mount immunofluorescence of Pax7+MyoD+ muscle progenitors (red), GFP+ wild-type PPF cells (green) and TUNEL+ apoptotic cells (blue) at E12.5. Scale bar, 100 μm. (MOV 1641 kb)

Early increase in apoptosis and defects in localization of muscle progenitors lead to CDH.

At E12.5, there is a marked increase in apoptotic cells and a decrease in costal muscle progenitors, and muscle progenitors are largely absent from regions with Gata4-null PPF cells in Prx1-creTg/+; Gata4fl/+; RosamTmG/+ diaphragms. Three-dimensional view of whole-mount immunofluorescence of Pax7+MyoD+ muscle progenitors (red), GFP+ Gata4-null PPF cells (green) and TUNEL+ apoptotic cells (blue) at E12.5. Scale bar, 100 μm. (MOV 1420 kb)

Costal muscle progenitors normally proliferate within the pleuroperitoneal folds.

At E12.5, myogenic progenitors proliferate at high levels and are intermingled with and surrounded by PPF cells in control Prx1-creTg/+; Gata4fl/+; RosamTmG/+ diaphragms. Three-dimensional view of whole-mount immunofluorescence of Pax7+MyoD+ muscle progenitors (red), GFP+ PPF cells (green) and EdU+ proliferative cells (blue) at E12.5. Scale bar, 100 μm. (MOV 2203 kb)

Early defects in proliferation and localization of muscle progenitors lead to CDH.

At E12.5, there is a marked decrease in proliferating cells and a decrease in costal muscle progenitors, and muscle progenitors are largely absent from regions with Gata4-null PPF cells in Prx1-creTg/+; Gata4fl/+; RosamTmG/+ diaphragms. Three-dimensional view of whole-mount immunofluorescence of Pax7+MyoD+ muscle progenitors (red), GFP+ Gata4-null PPF cells (green) and EdU+ proliferative cells (blue) at E12.5. Scale bar, 100 μm. (MOV 1391 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Merrell, A., Ellis, B., Fox, Z. et al. Muscle connective tissue controls development of the diaphragm and is a source of congenital diaphragmatic hernias. Nat Genet 47, 496–504 (2015). https://doi.org/10.1038/ng.3250

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng.3250

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing