Endocytic Protein Defects in the Neural Crest Cell Lineage and Its Pathway Are Associated with Congenital Heart Defects

Abstract

Endocytic trafficking is an under-appreciated pathway in cardiac development. Several genes related to endocytic trafficking have been uncovered in a mutagenic ENU screen, in which mutations led to congenital heart defects (CHDs). In this article, we review the relationship between these genes (including LRP1 and LRP2) and cardiac neural crest cells (CNCCs) during cardiac development. Mice with an ENU-induced Lrp1 mutation exhibit a spectrum of CHDs. Conditional deletion using a floxed Lrp1 allele with different Cre drivers showed that targeting neural crest cells with Wnt1-Cre expression replicated the full cardiac phenotypes of the ENU-induced Lrp1 mutation. In addition, LRP1 function in CNCCs is required for normal OFT lengthening and survival/expansion of the cushion mesenchyme, with other cell lineages along the NCC migratory path playing an additional role. Mice with an ENU-induced and targeted Lrp2 mutation demonstrated the cardiac phenotype of common arterial trunk (CAT). Although there is no impact on CNCCs in Lrp2 mutants, the loss of LRP2 results in the depletion of sonic hedgehog (SHH)-dependent cells in the second heart field. SHH is known to be crucial for CNCC survival and proliferation, which suggests LRP2 has a non-autonomous role in CNCCs. In this article, other endocytic trafficking proteins that are associated with CHDs that may play roles in the NCC pathway during development, such as AP1B1, AP2B1, FUZ, MYH10, and HECTD1, are reviewed.

Keywords: LRP1; LRP2; common arterial trunk; congenital heart defect; double outlet right ventricle; endocytic vesicle trafficking protein; neural crest cell.

Figure 1

Conditional Cre deletion of the floxed Lrp1 allele. Conditional Lrp1 knockout in different cell lineages during cardiac development was carried out using different Cre drivers. (a) Pie charts illustrating the percentages of different cardiac phenotypes and Lrp1^m/m cardiac phenotypes. (b) Lrp1 expression is illustrated in yellow in developing heart around E10.5–E11.5. Targeting Lrp1 deletion in neural crest cells using Wnt1-Cre (Wnt1^+/Cre: Lrp1^f/f) recapitulates the cardiac phenotypes of Lrp1^m/m mutants, which is illustrated in dotted green arrows. The ablation of LRP1 expression using Nkx2-5-Cre (Nkx2-5^+/Cre: Lrp1^f/f) is illustrated in pink hash, specifically in the PM, PE, AHF, and AV canal cushion. The ablation of LRP1 expression using Tie2-Cre (Tie2^+/Cre: Lrp1^f/f) is illustrated in blue hash and blue dots. The ablation of Lrp1 in double knockout of Nkx2-5-Cre and Tie2-Cre (Nkx2-5^+/Cre Tie2^+/Cre: Lrp1^f/f) is illustrated in pink and purple hash. The combined deletion of Lrp1 mediated by Tie2-Cre and Nkx2-5-Cre together demonstrated the high penetrance of the DORV/AVSD phenotype and increased the penetrance of AVSD. The ablation of Lrp1 using Mef2c-AHF-Cre (Mef2c-AHF^+/cre: Lrp1^f/f) is illustrated in orange hash. The ablation of Lrp1 using Twist2-Cre (Twist2^+/cre: Lrp1^f/f) is illustrated in navy blue. There is no cardiac phenotype of Nfatc^+/cre: Lrp1^f/f; the ablation of LRP1 expression is expressed in red. AHF: anterior heart field; AO: aorta; AS: aortic sac; AVC: atrioventricular canal; AVSD: atrioventricular septal defect; CNCC: cardiac neural crest cells; DORV: double outlet right ventricle; LA: left atrium; LV: left ventricle, OFT: outflow tract; PA: pulmonary artery; PE: pharyngeal endoderm; PM: pharyngeal mesoderm; RA: right atrium; RV: right ventricle; VSD: ventricular septum defect. Scale bars: 0.5 mm (modified from reference [27] Figure 3, with additional data added).

Figure 2

Schematic representation of the interaction of endocytic proteins that are associated with congenital heart defects. HECTD1 is involved in the retinoic acid pathway (blue); LRP2 and FUZ are involved in the hedgehog signaling pathway (red); FUZ is involved in the Wnt signaling pathway (green). TGN: trans-Golgi network; Ub: ubiquitin; MVB: multivesicular bodies; RSG1: RAB-like small GTPase 1. Modified from reference [20], Figure 2.