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

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.

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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.

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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.

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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.

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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. (di) 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. (bg) Section immunofluorescence. Scale bars, 1 mm (a), 50 μm (b,c), 100 μm (di), 20 μm (insets in di).

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

(al) Cdk4 (af) and cyclin D2 (gl) are downregulated in Gata4-null PPF cells. E12.5 section immunofluorescence. Scale bars, 100 μm (al), 20 μm (insets in al).

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)

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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

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