Disrupted ER membrane protein complex-mediated topogenesis drives congenital neural crest defects

Abstract

Multipass membrane proteins have a myriad of functions, including transduction of cell-cell signals, ion transport, and photoreception. Insertion of these proteins into the membrane depends on the endoplasmic reticulum (ER) membrane protein complex (EMC). Recently, birth defects have been observed in patients with variants in the gene encoding a member of this complex, EMC1. Patient phenotypes include congenital heart disease, craniofacial malformations, and neurodevelopmental disease. However, a molecular connection between EMC1 and these birth defects is lacking. Using Xenopus, we identified defects in neural crest cells (NCCs) upon emc1 depletion. We then used unbiased proteomics and discovered a critical role for emc1 in WNT signaling. Consistent with this, readouts of WNT signaling and Frizzled (Fzd) levels were reduced in emc1-depleted embryos, while NCC defects could be rescued with β-catenin. Interestingly, other transmembrane proteins were mislocalized upon emc1 depletion, providing insight into additional patient phenotypes. To translate our findings back to humans, we found that EMC1 was necessary for human NCC development in vitro. Finally, we tested patient variants in our Xenopus model and found the majority to be loss-of-function alleles. Our findings define molecular mechanisms whereby EMC1 dysfunction causes disease phenotypes through dysfunctional multipass membrane protein topogenesis.

Keywords: Cardiovascular disease; Development; Embryonic development; Genetics; Monogenic diseases.

Figure 1. Phenotypic assessment of Emc1 loss of function in Xenopus reveals craniofacial and cardiac dysmorphology.

1-cell–stage embryos were injected with either standard control MO or emc1 MO and phenotypically assessed at stage 45. (A) Representative images and measurements of 3 replicates of stage 45 control MO (n = 19) and emc1 MO (n = 21) embryo outflow tract morphology imaged with OCT imaging (dotted yellow line indicates measured diameter). Scale bar: 100 μm. (B) Representative images and percentages of 3 replicates of stage 45 control MO (n = 62) and emc1 MO (n = 55) embryo craniofacial cartilage stained with Alcian blue. Scale bar: 250 μm. (C) Immunoblot of pooled (n = 20) Emc1 protein in control and emc1 knockout/knockdown embryos. (D) Immunoblot of pooled (n = 20) Emc1 protein in emc1 knockdown and EMC1 rescued emc1 knockdown embryos. ****P < 0.0001, ***P < 0.0005 by (A) Student’s t test or (B) Fisher’s exact test. Bars indicate mean and SD.

Figure 2. emc1 depletion results in neural crest gene expression abnormalities early in development.

Embryos were injected into 1 cell at the 2-cell stage with emc1 MO followed by interrogation of neural crest markers via WISH. (A) WISH for markers of NCC cell lineage revealed that expression of earlier markers (pax3, snai2, and sox9) was present at expected developmental stages (stages 16 and 20 shown) but displayed abnormal distribution; the later marker sox10 was almost entirely lost (n = 45 per marker per stage done in 3 replicates; injected halves of embryos indicated by asterisks). Scale bar: 500 μm. (B) Schematic of the experimental setup, in which injection of MO into 1 cell of a 2-cell embryo allowed for 1 side of the embryo to develop under the effects of the MO injection; the other half served as an internal control for developmental phenotypes. (C) Markers showed abnormal distribution at later stages (stage 24 shown), suggesting mispatterning of the embryonic rostrum (n = 45 per marker per stage done in 3 replicates). Scale bar: 500 μm.

Figure 3. Proteomic analysis of emc1 knockdown uncovers affected developmental pathways including WNT signaling.

One-cell–stage embryos were injected with either standard control MO or emc1 MO, and LFQMS was carried out on stage 24 embryos. (A) Plot of mean protein levels from 3 biological replicates of 20 pooled emc1 morphants compared with control morphants as determined via LFQMS (statistically significantly increased proteins are shown in green, statistically significantly decreased proteins are shown in red, Fzd2 is shown as a black dot since it was only identified by one unique peptide sequence as opposed to the other proteins in the graph). (B) Plot of gene ontology terms for statistically significantly decreased proteins with human homologs displays enrichment for a subset of signaling pathways. (C) Representative image and quantitation of WNT signaling visualized in a transgenic Xenopus WNT reporter line as a comparison between mean fluorescence of emc1 MO-injected versus uninjected sides of the neural tube (white dotted line shows outline of neural tube and division between injected and uninjected sides). P, posterior; A, anterior; R, right; L, left. Scale bar: 100 μm. (D) Representative image and quantitation of β-catenin subcellular localization visualized in Xenopus neural tubes as a comparison between emc1 MO-injected versus control MO-injected embryos normalized to NLS-mCherry localization. Statistically significant protein changes assessed via ANOVA (A) with red and green points indicating P < 0.05. Scale bar: 10 μm. ****P < 0.0001, *P < 0.05 by Student’s paired t test (C) and Student’s t test (D). Bars indicate mean and SD.

Figure 4. Knockdown of EMC1 in human RPE cells and Xenopus affects multiple transmembrane proteins and embryo motility and activates ER stress.

(A) Immunofluorescence antibody labeling of EMC1 revealed a decrease in its expression after EMC1 siRNA treatment as compared with control siRNA treatments. Multipass membrane proteins (RHODOPSIN, nAChR, FZD2, FZD7) were abnormally localized (n = 10 high power fields per marker per condition done in 3 replicates). Scale bar: 20 μm. (B) Sample traces and measurement of control morphant (n = 30) and emc1 morphant (n = 30) tadpole movement over 10 seconds after stimulation (different colors differentiate distinct tadpoles) over 3 replicates. (C) Labeling of nAChR in the proximal tail of emc1-depleted stage 45 tadpoles showed sparse and less intense expression as compared with control counterparts. Scale bar: 50 μm. (D) Splicing assay for xbp1 in pooled (n = 30 per condition) stage 24 Xenopus embryos displayed increased splicing with emc1 MO depletion compared with control embryos repeated in 4 biological replicates. Tunicamycin treatment acted as positive control. (E) Immunoblotting for Fzd7 showed similar levels of Fzd7 in pooled (n = 30 per stage per condition) emc1 morphants as compared with control morphants at stage 14, but a marked decrease in levels at stage 24. ***P < 0.0005 by Student’s t test. Bars indicate mean and SD.

Figure 5. Neural crest marker expression rescued in emc1 morphants by modulating β-catenin.

Expression of sox10 in control MO-injected embryos raised in media containing DMSO showed stereotyped expression of this marker at stage 20; loss of sox10 expression in stage 20 embryos was observed after injection with emc1 MO. sox10 expression was partially rescued by injection of NLS-fused β-catenin mRNA or the small molecular GSK3β inhibitor CHIR 99021. Three replicates of 15 embryos were analyzed for each condition. Scale bar: 500 μm.

Figure 6. Effects of EMC1 depletion in human embryonic stem cell –derived NCCs.

(A) Schematic of the induction protocol used to generate hNCCs in which hESCs were exposed to CHIR 99021 for 2 days in serum-free medium prior to withdrawal of this agent and continued culture for 3 additional days. (B) Immunofluorescent antibody staining for NCC markers PAX7 and SOX10 at day 5 in EMC1 siRNA-treated cells as compared with control siRNA-treated cells performed in 2 separate biological replicates. Scale bar: 100 μm. (C) qPCR analysis at day 2 and day 5 showed a decrease in transcripts of EMC1 performed in 2 separate biological replicates. (D) qPCR analysis at day 5 performed in 2 separate biological replicates showed decreased levels of several NCC marker transcripts (PAX7, SNAI2, SOX9, FOXD3, SOX10), although other markers appeared to be unaffected (PAX3). (E) Quantified percentage of cells expressing each PAX7 and SOX10 protein performed in 2 separate biological replicates.

Figure 7. Xenopus model of emc1 loss of function provides a platform for testing patient variant pathogenicity.

Injection of wild-type EMC1 mRNA and to a lesser extent p.Gly471Arg (c.1411G>C) variant mRNA restored sox10 expression and tadpole movement in emc1-depleted embryos, whereas the mRNA of all other variants did not. (A) Representative images and percentages of embryos with sox10 expression after injection with emc1 MO at the 1-cell stage followed by injection with wild-type (n = 31) or variant EMC1 mRNA (n = 31 for Y23*, 29 for T82M, 20 for R105*, 31 for A144T, 30 for R404*, 29 for G471R, 28 for G868R, 25 for P874Rfs, and 31 for R881C) in 1 cell of the 2-cell stage over 3 biological replicates (injected halves of embryos are indicated by asterisks). Scale bar: 500 μm. (B) Schematic diagram of EMC1 protein with domain annotations and locations and effects of patient variants. CHD, congenital heart disease; GDD, global developmental delay; VIS, vision impairment; PQQ2, pyrrolo-quinoline quinone redox coenzyme domain; DUF, domain of unknown function. (C) Measurement of tadpole motility after coinjection with emc1 MO and wild-type (n = 15) or variant EMC1 mRNA (n = 15 for Y23*, 15 for T82M, 9 for R105*, 9 for A144T, 9 for R404*, 8 for G471R, 10 for G868R, 16 for P874Rfs, and 12 for R881C) at the 1 cell stage over 3 biological replicates. ANOVA P values were calculated as a comparison of mock injection to all other groups (A) and MO only to all other groups (C). ****P < 0.0001, ***P < 0.0005, **P < 0.005, *P < 0.05 by post hoc Tukey’s test of multiple comparisons of mock injection to all other groups (A) and MO only to all other emc1 MO-injected groups (C). Bars indicate mean and SD.