XB-ART-56617J Clin Invest February 3, 2020; 130 (2): 813-826.
Disrupted ER membrane protein complex-mediated topogenesis drives congenital neural crest defects.
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.
PubMed ID: 31904590
PMC ID: PMC6994125
Article link: J Clin Invest
Genes referenced: chrd.1 foxd3 fzd2 fzd7 nup85 pax7 rpe slc12a3 snai2 sox10 sox9 xbp1
GO keywords: neural crest cell development
Morpholinos: emc1 MO1
Disease Ontology terms: retinitis pigmentosa
Article Images: [+] show captions
|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 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.|
|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 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.|