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Gata-6 expression is decreased in diaphragmatic and pulmonary mesenchyme of fetal rats with nitrofen-induced congenital diaphragmatic hernia

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Abstract

Purpose

Congenital diaphragmatic hernia (CDH) and associated pulmonary hypoplasia are thought to be caused by a malformation of the underlying diaphragmatic and airway mesenchyme. GATA binding protein 6 (Gata-6) is a zinc finger-containing transcription factor that plays a crucial role during diaphragm and lung development. In the primordial diaphragm, Gata-6 expression is restricted to mesenchymal compartments of the pleuroperitoneal folds (PPFs). In addition, Gata-6 is essential for airway branching morphogenesis through upregulation of mesenchymal signaling. Recently, mutations in Gata-6 have been linked to human CDH. We hypothesized that diaphragmatic and pulmonary Gata-6 expression is decreased in the nitrofen-induced CDH model.

Methods

Time-mated rats were exposed to either nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms (n = 72) and lungs (n = 48) were microdissected on selected timepoints D13, D15 and D18, and divided into control and nitrofen-exposed specimens (n = 12 per sample, timepoint and experimental group, respectively). Diaphragmatic and pulmonary gene expression of Gata-6 was analyzed by qRT-PCR. Immunofluorescence-double staining for Gata-6 was combined with the diaphragmatic mesenchymal marker Gata-4 and the pulmonary mesenchymal marker Fgf-10 to evaluate protein expression and localization in fetal diaphragms and lungs.

Results

Relative mRNA expression levels of Gata-6 were significantly decreased in PPFs on D13 (0.57 ± 0.21 vs. 2.27 ± 1.30; p < 0.05), developing diaphragms (0.94 ± 0.59 vs. 2.28 ± 1.89; p < 0.05) and lungs (0.56 ± 0.16 vs. 0.71 ± 0.39; p < 0.05) on D15 and fully muscularized diaphragms (1.20 ± 1.10 vs. 2.52 ± 1.86; p < 0.05) and differentiated lungs (0.56 ± 0.05 vs. 0.77 ± 0.14; p < 0.05) on D18 of nitrofen-exposed fetuses compared to controls. Confocal laser scanning microscopy demonstrated markedly diminished immunofluorescence of Gata-6 mainly in diaphragmatic and pulmonary mesenchyme, which was associated with a reduction of proliferating mesenchymal cells in nitrofen-exposed fetuses on D13, D15, and D18 compared to controls.

Conclusion

Decreased Gata-6 expression during diaphragmatic development and lung branching morphogenesis may disrupt mesenchymal cell proliferation, causing malformed PPFs and reduced airway branching, thus leading to diaphragmatic defects and pulmonary hypoplasia in the nitrofen-induced CDH model.

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Authors and Affiliations

  1. National Children’s Research Centre, Our Lady’s Children’s Hospital, Crumlin, Dublin 12, Ireland

    Toshiaki Takahashi, Florian Friedmacher, Julia Zimmer & Prem Puri

  2. Conway Institute of Biomolecular and Biomedical Research, School of Medicine and Medical Science, University College Dublin, Dublin, Ireland

    Prem Puri

Authors
  1. Toshiaki Takahashi
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  2. Florian Friedmacher
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  3. Julia Zimmer
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  4. Prem Puri
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Correspondence to Prem Puri.

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This research was supported by the National Children’s Research Centre and the Children’s Medical and Research Foundation, Ireland.

Conflict of interest

The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.

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Takahashi, T., Friedmacher, F., Zimmer, J. et al. Gata-6 expression is decreased in diaphragmatic and pulmonary mesenchyme of fetal rats with nitrofen-induced congenital diaphragmatic hernia. Pediatr Surg Int 34, 315–321 (2018). https://doi.org/10.1007/s00383-017-4219-8

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  • DOI: https://doi.org/10.1007/s00383-017-4219-8

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