Marquez J et al. (2020), Disrupted ER membrane protein complex-mediated ...

XB-ART-56617

J Clin Invest 2020 Feb 03;1302:813-826. doi: 10.1172/JCI129308.

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

Marquez J , Criscione J , Charney RM , Prasad MS , Hwang WY , Mis EK , García-Castro MI , Khokha MK .

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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.

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Species referenced: Xenopus
Genes referenced: chrd ctnnb1 foxd3 fzd2 fzd7 nup85 pax7 rpe slc12a3 snai2 sox10 sox9 xbp1
GO keywords: neural crest cell development
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	Graphical Abstract
	Figure 1. 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. 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. 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. (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. 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. (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. 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 < 0.005, *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.
	Supplemental Figure 1 Knockdown of emc1 causes abnormal pigment cell morphology. Representative images of control MO and emc1 MO injected stage 45 embryo pigment cell morphology. 30 embryos were imaged for each condition over 3 biological replicates. Scale bar indicates 500 μm
	Supplemental Figure 2 F0 CRISPR mosaic knockout of emc1 causes abnormal craniofacial morphology. (A) Representative images and quantitation of Cas9 only (n=48) and Cas9 + emc1 sgRNA injected (n=60) stage 45 embryos stained with Alcian blue over 3 biological replicates. Scale bar indicates 250 μm. (B) Example TIDE analysis of insertion and deletion sizes along with their predicted effects in one embryo. This analysis of single embryos was carried out in 5 replicates to ensure consistent CRISPR mediated targeting. “Other” indicates changes that could not be analyzed via TIDE due to large size of indel. ****p < 0.0001 by Fisher’s exact test. Bars indicate mean and SD.
	Supplemental Figure 3 Fragment analysis of xbp1 splicing demonstrates increased endoplasmic reticulum stress in emc1 morphants. (A) Examples of traces obtained from fragment analysis for xbp1 spliced and un-spliced forms in 4-5 replicates of 30 pooled stage 24 control embryos, tunicamycin (positive control) treated embryos, and emc1 morphant embryos. (B) Ratios of area under the curve measurements corresponding to peaks of spliced to unspliced forms of xbp1 in stage 24 control embryos, tunicamycin (positive control) treated embryos, and emc1 morphant embryos demonstrate an increase in xbp1 splicing in emc1 morphants. *p<0.05 by Student’s T-test. Bars indicate mean and SD.
	Supplemental Figure 4 TUNEL staining for cell death of St 20 embryos injected with MO in one cell at the two-cell stage. Representative images of embryos injected with control, emc1, or nup85 MO affecting half of the embryo (indicated by asterisk side). Three replicates of 10 embryos were analyzed for each condition. Scale bar indicates 500 μm.
	Supplemental Figure 5 Immunoblot analysis of FZD2 levels reveals proteasomal degradation as the source of FZD2 clearance in EMC1 depleted RPE cells. (A) Immunoblot of FZD2 in cells transfected with a control siRNA and subjected to either cycloheximide treatment alone or cycloheximide and MG132 proteasomal inhibition performed in 3 biological replicates. (B) Immunoblot of FZD2 in cells transfected with EMC1 siRNA and subjected to either cycloheximide treatment alone or cycloheximide and MG132 proteasomal inhibition performed in 3 biological replicates. (C) Normalized densitometry of FZD2 levels from immunoblot assays. Bars indicate mean and SD.
	Supplemental Figure 6 EMC1 expression during early human neural crest cell induction. qPCR analysis of EMC1 transcripts during the human NCC induction protocol shows a sustained level of EMC1 transcripts within one day after beginning induction for 2 biological replicates at each time point.
	Supplemental Figure 7 EMC1 knockdown via siRNA results in decreased FZD7 in hESC derived neural crest cells. (A) Immunofluorescence antibody labeling of EMC1 and FZD7 at day 2 of neural crest induction reveals a decrease in both as well as a more punctate appearance in residual FZD7 signal as compared to control siRNA treated cells. Cells in 3 replicates of 2-3 high power fields were assessed for each marker per condition. Scale bar indicates 5 μm. (B) Immunofluorescence antibody labeling of EMC1 and FZD7 at day 5 of neural crest induction reveals a recovery in EMC1 while FZD7 remains diminished and in a punctate pattern. Cells in 3 replicates of 2-4 high power fields were assessed for each marker per condition. Scale bar indicates 5 μm. Bars indicate mean and SD
	Supplemental Figure 8 Knockdown of emc1 in Xenopus results in abnormal nAChR signal in tail neuromuscular junctions that can be rescued through exogenous introduction of human EMC1. Injection of wildtype EMC1 mRNA and to a lesser extent p.Gly471Arg (c.1411G>C) variant mRNA restores nAChR patterns of expression in emc1 depleted tail neuromuscular tissue while mRNA of other variants do not. Three replicates of 8 embryos were analyzed for each condition. Scale bar indicates 50 μm. Images of uninjected control and emc1 MO labeling of nAChR are from subregions of images shown in Figure 4C.

References [+] :

Abbruzzese, The Wnt receptor Frizzled-4 modulates ADAM13 metalloprotease activity. 2015, Pubmed, Xenbase