Loss of microRNAs in neural crest leads to cardiovascular syndromes resembling human congenital heart defects

Abstract

Objective: Congenital heart defects represent the most common human birth defects. Even though the genetic cause of these syndromes has been linked to candidate genes, the underlying molecular mechanisms are still largely unknown. Disturbance of neural crest cell (NCC) migration into the derivatives of the pharyngeal arches and pouches can account for many of the developmental defects. The goal of this study was to investigate the function of microRNA (miRNA) in NCCs and the cardiovascular system.

Methods and results: We deleted Dicer from the NCC lineage and showed that Dicer conditional mutants exhibit severe defects in multiple craniofacial and cardiovascular structures, many of which are observed in human neuro-craniofacial-cardiac syndrome patients. We found that cranial NCCs require Dicer for their survival and that deletion of Dicer led to massive cell death and complete loss of NCC-derived craniofacial structures. In contrast, Dicer and miRNAs were not essential for the survival of cardiac NCCs. However, the migration and patterning of these cells were impaired in Dicer knockout mice, resulting in a spectrum of cardiovascular abnormalities, including type B interrupted aortic arch, double-outlet right ventricle, and ventricular septal defect. We showed that Dicer loss of function was, at least in part, mediated by miRNA-21 (miR-21) and miRNA-181a (miR-181a), which in turn repressed the protein level of Sprouty 2, an inhibitor of Erk1/2 signaling.

Conclusions: Our results uncovered a central role for Dicer and miRNAs in NCC survival, migration, and patterning in craniofacial and cardiovascular development which, when mutated, lead to congenital neuro-craniofacial-cardiac defects.

Figure 1. Neural-crest-specific ablation of Dicer resulted in severe craniofacial defects

In all images, wild type (+/+) control and NCC-Dicer mutant (−/−) embryos are shown. (A) Gross morphology of E17.5 embryos. Neural-crest-specific Dicer mutant embryos displayed severe craniofacial defects (arrows). (B) Skeletal preparation of E17.5 embryos. Most of the neural-crest-derived cartilages and bones, including maxilla, mandible and nasal bones (arrows), were absent in Dicer mutants. (C) Thoracotomies showed the hypoplasia of thymus (arrows) in mutant embryos. (D) Dicer mutant mice exhibited underdeveloped thyroid glands (arrows). (E) TUNEL assay showed an abnormal apoptosis in the 1st (1PA) and 2nd (2PA) pharyngeal arches in mutant embryos. (F) Statistics of TUNEL-positive cells. Three discontinuous sections containing 1st and 2nd pharyngeal arches were stained and counted. (G) Whole mount NBS staining consistently revealed the abnormal apoptosis in mutants. (H) The morphology of 1st and 2nd pharyngeal arches of E10.5 embryos showed the reduced size of pharyngeal arches in Dicer mutants. (I–K) Whole mount β-gal staining of E11.5 and E13.5 embryos containing Rosa-LacZ indicator. E11.5 Dicer mutant embryos failed to form the lateral lingual swellings (I, arrows); E13.5 Dicer mutant embryos displayed a cleft lip (J, arrows) and a hypoplastic pinna (K, arrows). (L) Histological examination of E13.5 embryonic heads. The neural-crest-derived Meckel’s cartilage, hyoid cartilage and tooth primordium failed to form in mutants, while the development of non-neural-crest-derived cartilage primordium of clivus is unaffected. C: cartilage primordium of clivus; H: hyoid cartilage; M: Meckel’s cartilage; T: tongue; TP: tooth primordium.

Figure 2. Neural-crest-specific ablation of Dicer led to multiple cardiovascular defects

(A) The penetrance of cardiovascular defects displayed in Dicer mutant mice. (B) Gross morphology of heart and great vessels from E17.5 embryos. The Dicer mutant mice exhibited a type B interrupted aortic arch (arrow). (C–D) Histological examination of E17.5 embryonic hearts. The mutant hearts possessed ventricular septal defect (C) accompanied with a double outlet right ventricle (D). (E) Great vessels shown by ink injection. The type B interrupted aortic arch was consistently found in mutant embryos. (F–G) Histological examination of defects in great vessels. A right subclavian artery was ectopically originated from the descending aorta (G) and extended rightward behind the esophagus (F, G). The asterisk in (G) indicates the interruption of aortic arch. AAo: ascending aorta; Ao: aorta; AoA: aortic arch; DAo: descending aorta; DOSV: double outlet right ventricle; E: esophagus; H: heart; IA: innominate artery; IAA: interrupted aortic arch; LA: left atrium; LCA: left carotid artery; LSA: left subclavian artery; LV: left ventricle; Pa: pulmonary artery; RA: right atrium; RCA: right carotid artery; RSA: right subclavian artery; RRSA: retroesophageal right carotid artery; RV: right ventricle; T: trachea; Th: thymus; VSD: ventricular septal defect.

Figure 3. Dicer and miRNAs are required for proper development of neural crest cells

(A) Whole mount β-gal staining of E11.5 embryos containing Rosa-LacZ indicator showed the migration of cardiac neural crest to the caudal limit of outflow tract (arrows). (B) Histological examination of the stained outflow tract. The Dicer-deficient cardiac NCCs failed to form two columns of condensed mesenchyme in outflow tract. (C) Histological examination of E11.5 heart. The entrance of outflow tract for future aorta (asterisks) had a rightward malalignment with muscular interventricular septum. (D) Whole mount β-gal staining of E13.5 embryonic hearts indicated the rotation defect and malalignment of conus cushion. Two prongs of Dicer-deficient cardiac NCCs (arrows) located in right ventricle in a side-by-side manner. The dash lines indicate the ventricular septum. (E) Neural-crest-derived great vessels in E13.5 embryos were shown by β-gal staining. The blue lines in cartoon indicate the neural-crest-derived vessels, while the red dash lines indicate the vessels not contributed by cardiac NCCs, which cannot be shown in β-gal staining. Mutant embryos displayed a hypoplastic aortic arch artery. Histological examination of aortic arch was performed in the orientation indicated by dashed lines. The regions (E’, E’’) indicated by arrows were shown at bottom right and Supplemental Figure VII. The aortic arch in mutant embryos had a thinner vessel wall with fewer smooth muscle cells. (F) TUNEL assay with E13.5 embryo sections containing aortic arch showed an abnormal apoptosis of neural-crest-derived smooth muscle cells in the vessels in Dicer mutant embryos. (G) Statistics of TUNEL-positive cells. Three discontinuous sections containing aortic arches were stained and counted. Ao: aorta; AoA: aortic arch; E: esophagus; H: heart; LA: left atrium; LCA: left carotid artery; LSA: left subclavian artery; LV: left ventricle; M: muscular interventricular septum; Pt: pulmonary trunk; RA: right atrium; RCA: right carotid artery; RSA: right subclavian artery; RV: right ventricle; T: trachea.

Figure 4. Dysregulation of gene expression of components in MEK/ERK signaling cascade in NCC-Dicer mutants

(A–B) Whole mount immunochemistry detecting phospho-Erk1/2 (pErk1/2) in E10.5 (A, side view) and E11.5 (B, frontal view) embryos. pErk1/2 was downregulated in neural-crest-derived pharyngeal arches but not in limb buds of Dicer mutant embryos. (C) Whole mount immunochemistry detecting Sprouty2 in E11.5 embryos. Sprouty2 was upregulated in neural-crest-derived pharyngeal arches of Dicer mutant embryos. (D) The dysregulation of pErk1/2 and Sprouty2 was shown by Western blot with pharyngeal arch tissue samples. (E–K) Examination of gene expression by immunohistochemistry detecting pErk1/2 (E), Sprouty1 (F), Sprouty2 (G), Crkl (H), SHP-2 (I), Sos1 (J) and pMEK1/2 (K) expression in first pharyngeal arches of E10.5 Dicer mutant embryos. 1PA: 1^st pharyngeal arch; 2PA: second pharyngeal arch; LB: limb bud.

Figure 5. Repression of sprouty expression by miRNAs

(A) Detection of the haploinsufficiency of mature miRNAs and the accumulation of miRNA precursors in mutant pharyngeal arch tissue samples by Northern blot. (B) Quantitative real-time PCR assays to measure the expression level of mature miR-181a and miR-21 in mutant pharyngeal arch tissues. *: P < 0.01. (C) Hela cells were transfected with indicated luciferase reporters, along with either miR-21 or miR-181a duplex mimic. A Renilla luciferase vector was cotransfected to serve as an internal control for normalization. Cells were harvested and luciferase activity measured 24 hours after transfection. Values are presented as mean luciferase activity ± SD relative to the luciferase activity of reporters with control duplex mimic. *: P < 0.05. (D) Dicer and miRNAs are suggested to participate in a regulatory cascade to modulate the expression levels and activities of MEK/ERK signaling pathway in neural crest cells.