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Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes
Published on: February 13, 2021
Intra-amniotic transient transduction of the periderm with a viral vector encoding TGFβ3 prevents cleft palate in
Chadwick Wu1, Masa Endo, Byung H Yang
1Division of Plastic and Reconstructive Surgery, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.
Insights
Intra-amniotic gene transfer of transforming growth factor β3 (TGFβ3) can prevent cleft palate in mouse models. This approach restored palatal fusion by targeting superficial cells, offering potential in utero therapy.
Area of Science:
- Developmental Biology
- Regenerative Medicine
- Gene Therapy
Background:
- Cleft palate is a congenital defect caused by failed embryonic palatal shelf fusion.
- Transforming growth factor β3 (TGFβ3) is crucial for palatal fusion during embryonic development (E13-E15 in mice).
- Tgfβ3-deficient mice exhibit a complete cleft palate phenotype, highlighting TGFβ3's essential role.
Purpose of the Study:
- To investigate the efficacy of intra-amniotic gene transfer for preventing cleft palate.
- To determine if restoring TGFβ3 expression can rescue palatal fusion in Tgfβ3(-/-) mouse models.
- To assess the therapeutic potential of in utero gene therapy for cleft palate.
Main Methods:
- Adenoviral vectors encoding Tgfβ3 were microinjected into the amniotic sacs of mouse embryos at various developmental stages.
- Tgfβ3(-/-) mouse model was used to study cleft palate development and treatment.
- Palatal fusion and mesenchymal confluence were assessed in transduced fetuses.
Main Results:
- Intra-amniotic Tgfβ3 gene transfer successfully restored palatal fusion in Tgfβ3(-/-) fetuses.
- High success rates (100% at E12.5 and E13.5, 82% at E14.5, 75% at E15.5) were observed depending on injection timing.
- Transduction of the superficial peridermal cell layer was sufficient to induce MEE fusion in a cell nonautonomous manner.
Conclusions:
- Intra-amniotic gene transfer of TGFβ3 is a viable strategy to prevent cleft palate in a mouse model.
- This approach demonstrates therapeutic potential for in utero treatment of cleft palate, particularly cases involving midline epithelial dysfunction.
- TGFβ3's role in palatal fusion can be therapeutically targeted via non-invasive gene delivery to embryonic tissues.
Abstract:
Cleft palate is a developmental defect resulting from the failure of embryonic palatal shelves to fuse with each other at a critical time. Immediately before and during palatal fusion (E13-E15 in mice), transforming growth factor β3 (TGFβ3) is expressed in the palatal shelf medial edge epithelium (MEE) and plays a pivotal role in palatal fusion. Using Tgfβ3(-/-) mice, which display complete penetrance of the cleft palate phenotype, we tested the hypothesis that intra-amniotic gene transfer could be used to prevent cleft palate formation by restoring palatal midline epithelial function. An adenoviral vector encoding Tgfβ3 was microinjected into the amniotic sacs of mouse embryos at successive developmental stages. Transduced Tgfβ3(-/-) fetuses showed efficient recovery of palatal fusion with mesenchymal confluence following injection at E12.5 (100%), E13.5 (100%), E14.5 (82%), and E15.5 (75%). Viral vectors injected into the amniotic sac transduced the most superficial and transient peridermal cell layer but not underlying basal epithelial cells. TGFβ3 transduction of the peridermdal cell layer was sufficient to induce adhesion, fusion, and disappearance of the palatal shelf MEE in a cell nonautonomous manner. We propose that intra-amniotic gene transfer approaches have therapeutic potential to prevent cleft palate in utero, especially those resulting from palatal midline epithelial dysfunction.
