Epithelial fusion underlies many vital organogenic processes during embryogenesis. Disruptions to these cause a significant number of human birth defects, including ocular coloboma. We provide robust spatial-temporal staging and unique anatomical detail of optic fissure closure (OFC) in the embryonic chick, including evidence for roles of apoptosis and epithelial remodelling. We performed complementary transcriptomic profiling and show that Netrin-1 (NTN1) is precisely expressed in the chick fissure margin at the fusion plate but is immediately downregulated after fusion. We further provide a combination of protein localisation and phenotypic evidence in chick, humans, mice and zebrafish that Netrin-1 has an evolutionarily conserved and essential requirement for OFC, and is likely to have an important role in palate fusion. Our data suggest that NTN1 is a strong candidate locus for human coloboma and other multi-system developmental fusion defects, and show that chick OFC is a powerful model for epithelial fusion research.
- Joe Rainger
- Joe Rainger
- Joe Rainger
- Joe Rainger
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: All animal work was carried out in strict accordance with the United Kingdom Home Office Animal (Scientific Procedures) Act 1986. All chicken experiments, breeding and care procedures were approved and carried out under license from the UK Home Office (PPL 7008940 - Prof Helen Sang) and subject to local ethical review by the Roslin Institute AWERB. No regulated procedures were used in this study. Generation and maintenance of memGFP flock were in accordance with annex III of Directive 2010/63 EU and Home Office Codes of Practice. All mouse and zebrafish work was conducted in compliance with protocols approved by the Institutional Animal Care and Use Committee at Harvard Medical School, and at The NIH National Eye Institute. Mice were used from an existing study (Yung et al., Development. 2015). Ntn -/- (Ntn1tm1.1Good, MGI:5888900) and C57Bl/6J animals were maintained on a standard 12hr light-dark cycle. Mice received food and water ad lib and were provided with fresh bedding and nesting daily. All experiments were conducted in agreement with the Animals (Scientific Procedures) Act 1986 and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Pregnant dams were anaesthetised by CO2 asphyxiation and euthanised by cervical dislocation. Embryos were collected at E11.5, E15.5 and E16.5. All embryos were immediately culled on ice by decapitation. All zebrafish embryos/larvae are taken at between 30 hpf-56 hpf and immediately anaesthetised with tricaine methane sulfonate (MS222, 168 mg/l) on ice. Embryos are then euthanised in bleach solution (sodium hypochlorite 6.15%) in water at 1 part bleach to 5 parts water. The larvae remain in this solution at least five minutes prior to disposal to ensure death.
Human subjects: Human foetal eyes were obtained from the Joint Medical Research Council UK (grant # G0700089)/Wellcome Trust (grant # GR082557) Human Developmental Biology Resource (http://www.hdbr.org/). The consent, use and disposal of HDBR samples is regulated by the UK Human Tissue Authority (HTA). The HDBR is a Research Ethics Committee (REC) approved and HTA licenced tissue bank. This means that most research projects based within the UK do not need to obtain their own REC approval.
- Jeremy Nathans, Johns Hopkins University School of Medicine, United States
- Received: November 23, 2018
- Accepted: June 3, 2019
- Accepted Manuscript published: June 4, 2019 (version 1)
© 2019, Hardy et al.
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