Identification of genetic suppressors for a BSCL2 lipodystrophy pathogenic variant in Caenorhabditis elegans

Abstract

Seipin (BSCL2), a conserved endoplasmic reticulum protein, plays a critical role in lipid droplet (LD) biogenesis and in regulating LD morphology, pathogenic variants of which are associated with Berardinelli-Seip congenital generalized lipodystrophy type 2 (BSCL2). To model BSCL2 disease, we generated an orthologous BSCL2 variant, seip-1(A185P), in Caenorhabditis elegans. In this study, we conducted an unbiased chemical mutagenesis screen to identify genetic suppressors that restore embryonic viability in the seip-1(A185P) mutant background. A total of five suppressor lines were isolated and recovered from the screen. The defective phenotypes of seip-1(A185P), including embryonic lethality and impaired eggshell formation, were significantly suppressed in each suppressor line. Two of the five suppressor lines also alleviated the enlarged LDs in the oocytes. We then mapped a suppressor candidate gene, lmbr-1, which is an ortholog of human limb development membrane protein 1 (LMBR1). The CRISPR/Cas9 edited lmbr-1 suppressor alleles, lmbr-1(S647F) and lmbr-1(P314L), both significantly suppressed embryonic lethality and defective eggshell formation in the seip-1(A185P) background. The newly identified suppressor lines offer valuable insights into potential genetic interactors and pathways that may regulate seipin in the lipodystrophy model.

Keywords: Caenorhabditis elegans; CRISPR/Cas9; Genetic suppressor; LMBR-1; Lipid droplet; Seipin.

Fig. 1.

Functional analysis of the A185P mutation in a seip-1::mScarlet reporter gene. (A) Schematic diagram of seip-1(A185P)::mScarlet. (B,C) Visualization of mScarlet fluorescence to assess SEIP-1 localization. Green arrows indicate the presence of multiple nuclei in the posterior cell. (D,E) DAPI staining of embryos to assess eggshell permeability. Yellow arrows indicate the presence of stained chromatin. (F,F′) Brood size and embryonic viability of the mScarlet reporter genes. ns, not significant; **P<0.005 (one-way ANOVA with post hoc test). (G) SEIP-1 localization and lipid droplets in the adult germline. Representative images of animals containing the seip-1::mScarlet (G, magenta) or seip-1(A185P)::mScarlet (H, magenta) reporter, as visualized using mScarlet protein fluorescence (G,H, magenta), and the lipid dye BODIPY (G,H, green). Yellow arrows indicate the presence of enlarged lipid droplets. Oocytes are numbered conventionally (−1, −2 and −3) relative to the spermatheca, which is the site of fertilization. Scale bars: 10 μm. Each experiment was repeated at least three times. n values are indicated in the graphs.

Fig. 2.

Forward genetic screen for suppressors of the embryonic lethality of a seipin patient-specific allele. (A) Diagram of the location of the seip-1(A185P) mutation in the seip-1 gene. (B) Strategy to identify genetic modifiers that restored embryonic viability in the seip-1(A185P) mutant. Created with BioRender.com. (C) Total brood size of each isolated suppressor line, wild type and seip-1(A185P) mutant over 60 h post mid-L4. (D) The percentage of embryonic viability was significantly restored in each suppressor line when compared with seip-1(A185P) mutant only. Data are mean±s.d. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test, **P<0.005. Each experiment was repeated at least three times. n values are indicated in the graphs.

Fig. 3.

Defective eggshell formation was restored in each suppressor line. (A,B) Schematic diagram of DAPI staining assay in C. elegans embryos to assess eggshell permeability. (C-H) Representative images of the DAPI staining assay showed only the first polar body stained in wild type (A,E, white arrows), whereas DAPI penetrated the eggshell to stain the zygotic chromatin in seip-1(A185P) mutants (F,H, yellow arrows). DIC images of embryos are shown in D and G. (I) Quantification of the embryos with DAPI staining in wild type, seip-1(A185P) only and all suppressor lines. Statistical significance was determined using a Chi-square test. **P<0.005. Each experiment was repeated at least three times. n values are indicated in each panel.

Fig. 4.

Enlarged lipid droplets were not correlatively restored in the suppressor lines. (A-G) BODIPY-stained lipid droplets (LDs) in the −1 to −3 oocytes of wild type (A), seip-1(A185P) only (B) and all five suppressor lines (C-G). (H) Quantification of the enlarged LDs (diameter >1.5 μm) inside the −1 to −3 oocytes of each stained genotype. Data are mean±s.d. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test. *P<0.05; **P<0.005. Scale bars: 10 μm. Each experiment was repeated for at least three times. n values are indicated in each panel.

Fig. 5.

Identification of the genetic modifiers by MIP-MAP genome mapping and whole-genome sequencing. (A) Schematic diagram of two suppressor alleles of lmbr-1 were generated in the wild-type background. (B,C) Total brood size and embryonic viability of each isolated wild type, seip-1(A185P), lmbr-1 suppressor alleles, lmbr-1 null mutant, and double mutants of seip-1(A185P) and lmbr-1 alleles. *P<0.05, **P<0.005 (χ² test) (D) DAPI penetration in the early embryos of each tested mutant and wild type.**P<0.005 (χ² test). (E-K) Representative images of the BODIPY-stained LDs in the −1 to −3 oocytes of wild type (E), seip-1(A185P) (F), lmbr-1(P314L) (I), lmbr-1(S647F) (J), lmbr-1Δ (K), and lmbr-1(P314L); seip-1(A185P) (G) and lmbr-1(S647F); seip-1(A185P) double mutants (H). (L) Quantification of the enlarged LDs (diameter >1.5 μm) in the indicated genotypes. *P<0.05, **P<0.005 (one-way ANOVA with Tukey's post hoc test). Each experiment was repeated for at least three times. n values are indicated in each panel.