Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Apr 1;26(7):1268-1279.
doi: 10.1093/hmg/ddx031.

Rdh10 loss-of-function and perturbed retinoid signaling underlies the etiology of choanal atresia

Hiroshi Kurosaka  1 , Qi Wang  1 , Lisa Sandell  2 , Takashi Yamashiro  1 , Paul A Trainor  3   4
Affiliations

Rdh10 loss-of-function and perturbed retinoid signaling underlies the etiology of choanal atresia

Hiroshi Kurosaka et al. Hum Mol Genet. .

Abstract

Craniofacial development is a complex process that involves sequential growth and fusion of the facial prominences. When these processes fail, congenital craniofacial anomalies can occur. For example, choanal atresia (CA) is a congenital craniofacial anomaly in which the connection between the nasal airway and nasopharynx is completely blocked. CA occurs in approximately 1/5000 live births and is a frequent component of congenital disorders such as CHARGE, Treacher Collins, Crouzon and Pfeiffer syndromes. However, the detailed cellular and molecular mechanisms underpinning the etiology and pathogenesis of CA remain elusive. In this study, we discovered that mice with mutations in retinol dehydrogenase 10 (Rdh10), which perturbs Vitamin A metabolism and retinoid signaling, exhibit fully penetrant CA. Interestingly, we demonstrate Rdh10 is specifically required in non-neural crest cells prior to E10.5 for proper choanae formation, and that in the absence of Rdh10, Fgf8 is ectopically expressed in the nasal fin. Furthermore, we found that defects in choanae development are associated with decreased cell proliferation and increased cell death in the epithelium of the developing nasal cavity, which retards invagination of the nasal cavity, and thus appears to contribute to the pathogenesis of CA. Taken together, our findings demonstrate that RDH10 is essential during the early stages of facial morphogenesis for the formation of a functional nasal airway, and furthermore establish Rdh10 mutant mice as an important model system to study CA.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Expression of Rdh10 and activity of RARE-LacZ during craniofacial development. (A,B,D) Ventral view of Rdh10 expression in serial embryonic stages in wild-type mice. (E,F,H) Activity of RARE-LacZ reporter during embryonic maxillary development. Red arrowheads indicate the position of primitive choanae in B and F, invaginating nasal cavity in D and H. (C and G) Frontal section of E11.5 control embryo showing expression of Rdh10 (C) and activity of RARE-LacZ (G). Red arrowhead indicates oronasal membrane in C and G, and black dotted line indicates the boundary of epithelial cells lining the nasal cavity. MNP, medial nasal process. LNP, lateral nasal process. MXP, maxillary process. MN, mandibular process. Scale bar = 200 μm.
Figure 2
Figure 2
Confirmation of reduction of Rdh10 mRNA in mice with conditional null allele. (A) Schematic drawing of the method used to generate the Rdh10 conditional null allele (modified from reference (18). (B–E) In situ hybridization of Rdh10 using an Exon2 specific riboprobe. E10.5 and E11.5 control embryos (B and C) compared with the same stage of Cre-ERT2; Rdh10flox/flox embryos (D and E) which were administered tamoxifen at E7.5. Red arrowhead indicates the lambdoidal region in B,D; and primitive choanae in C,E. TMX E7.5, tamoxifen was administered at E7.5. E, eye. MXP, maxillary process. MN, mandibular process. MNP, medial nasal process. LNP, lateral nasal process. FL, forelimb. Scale bar = 500 μm in B,D and 200 μm in C,E.
Figure 3
Figure 3
Morphological and histological analysis of Rdh10 mutant maxilla. Nuclear fluorescent staining of control (A,B) and Rdh10trex (E), and Cre-ERT2; Rdh10flox/flox mice to which tamoxifen was administered at E7.5 (F). Red arrowhead indicates the position of primitive choana(e) in (A and B), and their absence (E and F). Yellow arrowhead in (F) shows partial cleft lip. Skeletal preparation of E15.5 control (C) and Cre-ERT2; Rdh10flox/flox embryos (G). Hematoxylin and eosin staining of frontal section of E15.5 control (D) and Cre-ERT2; Rdh10flox/flox (H) embryos. Yellow asterisk demarcates the premaxillary bone. TMX E7.5, administered tamoxifen at E7.5. MNP, medial nasal process. LNP, lateral nasal process. MXP, maxillary process. MN, mandibular process. PMX, premaxillary bone. MX, maxillary bone. P, palatine bone. NS, nasal septum. Scale bar = 200 μm in A,E and 500 μm in B-D, F-H.
Figure 4
Figure 4
Excision of Rdh10 resulted in elevation of Fgf8 expression and altered cell proliferation and cell death. (A–D) In situ hybridization for Fgf8 in E11.5 embryos. (B and D) Frontal section of E11.5 embryos hybridized with an Fgf8 riboprobe. Red arrowheads indicate the nasal fin. (E and F) Immunohistochemistry of frontal sections from E11.5 embryos. Proliferating cells are stained red and cells undergoing cell death are stained green (E and F). White arrowheads indicate proliferating cells and white arrows indicate TUNEL-positive cell death in the nasal fin. Dotted line indicates the extent of the nasal epithelium. (G) Statistical analysis showed significant reduction in number of proliferating cell and elevation of cell death in Cre-ERT2; Rdh10flox/flox nasal fin. TMX E7.5, administered tamoxifen at E7.5. MNP, medial nasal process. LNP, lateral nasal process. N, nasal cavity. PHH3, phosphohistone H3. C, control mice. M, mutant (Cre-ERT2; Rdh10flox/flox) mice. *P < 0.05.
Figure 5
Figure 5
Morphological analysis following conditional deletion of Rdh10 during craniofacial development. Nuclear fluorescent image of the maxillary complex in E11.5 control mouse embryos (A). Red arrowhead indicates the position of the primitive choana, which still forms in Wnt1Cre;Rdh10flox/flox (neural crest-specific deletion of Rdh10) mice (B). At E12.5, the nasal cavity invaginates posteriorly in control mice (C, red arrowhead), but is impaired and hypoplastic in E10.5 tamoxifen (TMX)-administered embryos (D, red arrowhead). (E) Schematic drawing of the phenotype of Cre-ERT2; Rdh10flox/flox embryos at E12.5 according to when the tamoxifen is administrated. TMX E10.5, administered tamoxifen at E10.5. MNP, medial nasal process. LNP, lateral nasal process. MXP, maxillary process.
Figure 6
Figure 6
All-trans retinal rescued CA in Rdh10trex/trex mutants. (A) Hematoxylin and eosin staining of frontal section of anterior position of E18.5 control mouse and (B) Rdh10trex mutant treated with retinal. At the posterior position, the nasal cavity and pharynx are clearly connected in the control (C, red arrowhead) and in the Rdh10trex mutant treated with retinal (D, red arrowhead).
Figure 7
Figure 7
Schematic drawing of the process of nasal cavity invagination and primitive choanae formation. Nasal cavity invagination takes place to connect the nasal cavity to the airway by rupturing the oronasal membrane during normal development (A). In Rdh10 mutants, the number of proliferating epithelial cells is reduced, and the nasal cavity cannot connect to the airway, resulting in CA (B).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Trainor P.A., Andrews B.T. (2013) Facial dysostoses, Etiology, pathogenesis and management. Am. J. Med. Genet. C. Semin. Med. Genet., 163C, 283–294. - PMC - PubMed
    1. Trainor P.A. (2010) Craniofacial birth defects, The role of neural crest cells in the etiology and pathogenesis of Treacher Collins syndrome and the potential for prevention. Am. J. Med. Genet. A, 152A, 2984–2994. - PMC - PubMed
    1. Kurosaka H., Iulianella A., Williams T., Trainor P.A. (2014) Disrupting hedgehog and WNT signaling interactions promotes cleft lip pathogenesis. J. Clin. Invest., 124, 1660–1671. - PMC - PubMed
    1. Dixon M.J., Marazita M.L., Beaty T.H., Murray J.C. (2011) Cleft lip and palate, understanding genetic and environmental influences. Nat. Rev. Genet., 12, 167–178. - PMC - PubMed
    1. Kwong K.M. (2015) Current Updates on Choanal Atresia. Front. Pediatr., 3, 52.. - PMC - PubMed

Publication types