Pathological ASXL1 Mutations and Protein Variants Impair Neural Crest Development

Abstract

The neural crest (NC) gives rise to a multitude of fetal tissues, and its misregulation is implicated in congenital malformations. Here, we investigated molecular mechanisms pertaining to NC-related symptoms in Bohring-Opitz syndrome (BOS), a developmental disorder linked to mutations in the Polycomb group factor Additional sex combs-like 1 (ASXL1). Genetically edited human pluripotent stem cell lines that were differentiated to NC progenitors and then xenotransplanted into chicken embryos demonstrated an impairment of NC delamination and emigration. Molecular analysis showed that ASXL1 mutations correlated with reduced activation of the transcription factor ZIC1 and the NC gene regulatory network. These findings were supported by differentiation experiments using BOS patient-derived induced pluripotent stem cell lines. Expression of truncated ASXL1 isoforms (amino acids 1-900) recapitulated the NC phenotypes in vitro and in ovo, raising the possibility that truncated ASXL1 variants contribute to BOS pathology. Collectively, we expand the understanding of truncated ASXL1 in BOS and in the human NC.

Keywords: ASXL1; Bohring-Opitz syndrome; Polycomb; ZIC1; neural crest.

Figure 1

Human Pluripotent Stem Cell Models for Bohring-Opitz Syndrome (A) Scheme illustrating the generation of human pluripotent stem cell lines carrying premature stop codon (PSC) mutations in ASXL1. (B) Scheme of human ASXL1 protein showing annotated domains (NR, nuclear receptor), and locations of mutations frequently reported in BOS patients (red tinted sector) and present in BOS-iPSC and ASXL1^PSC/PSC/ASXL1^PSC/+ hESC clones. (C) Expression of ASXL1 in BOS-iPSC lines and ASXL1^PSC/PSC/ASXL1^PSC/+ hESC clones relative to the respective iPSC/hESC control lines using primers targeting exon 4 (mean ± SEM, n ≥ 3 different clones/passages). (D) Sequences of reverse transcribed ASXL1 transcripts from ASXL1 mutant lines. (E and F) Representative blotting (n = 3–5 independent experiments) of ASXL1, using a monoclonal antibody raised against the N terminus, in an hESC line overexpressing a truncated ASXL1 variant (PB-ASXL1^PSC) (E) and in human iPSC and hESC lines (F). See also Figure S1.

Figure 2

ASXL1 Mutations Impair Differentiation of Neural Crest Progenitors (A) Timeline of neural crest (NC) differentiation protocol and bright-field images during in vitro differentiation of ASXL1^+/+ hESCs. Scale bar, 500 μm. (B) Percentage of attached neurospheres with emigrating cells at day 7 of NC differentiation of BOS-iPSC lines and ASXL1^PSC/PSC/ASXL1^PSC/+ clones (mean ± SEM, n = 3–13 different passages/clones and independent experiments, all relative to respective control lines; ^∗∗p<0.01). (C) Representative bright-field images of neurospheres at day 7 of NC differentiation. Scale bar, 100 μm. (D) Workflow of neurosphere transplantation experiments and representative bright-field and GFP images of chicken embryos directly (upper panel) and 48 h (lower panel) after transplantation of neurospheres (white arrows: exemplary transplanted GFP-ASXL1^+/+ neurosphere; dorsal view). Scale bar, 500 μm (chicken embryo) and 200 μm (neurospheres). (E) Quantification of migrating cells from experiments in (D); ASXL1^+/+, n = 9; ASXL1^PSC/PSC, n = 21 embryos (mean ± SEM, ^∗p = 0.037). Single dots indicate outliers. See also Figure S2.

Figure 3

Misregulation of Gene Networks Associated with NC Development and BOS Symptoms in ASXL1 Mutant Lines (A) Volcano plot exhibiting differentially expressed genes in ASXL1^PSC/PSC compared with ASXL1^+/+ day 7 NC progenitors (total mRNA sequencing; n = 3 ASXL1^+/+ and n = 7 ASXL1^PSC/PSC samples, different clones and independent differentiation experiments). (B) Most significant Medical Subject Headings (MeSH) terms associated with downregulated gene sets (p_adj < 0.05) in (A). (C) Diagram of genes with annotated functions in NC development and their degree of misregulation (p_adj < 0.05), based on (A). Diff., terminal differentiation. (D and E) Relative expression levels of canonical NC genes in day 7 NC differentiation cultures of ASXL1^PSC hESC lines (D) and BOS-iPSC lines (E) compared with respective control lines (mean ± SEM, n = 3–6 different clones/passages; NR2F1 in iPSC control, n = 2 different passages; ^∗p < 0.05). (F) Quantification of ZIC1 protein levels in NC cultures (day 7) derived from cell lines as in (D and E) compared with the respective control lines (based on western blotting, n = 3–4 different clones/passages, mean ± SEM, ^∗p < 0.05). (G and H) Rescue of the NC differentiation defect by ectopic expression of ZIC1. (G) Detection of ZIC1 overexpression in DOX-treated PB-ZIC1-ASXL1^+/+ by western blotting. (H) Analysis of attached neurospheres with emigrating cells in PB-ZIC1-ASXL1^+/+ and PB-ZIC1-ASXL1^PSC/PSC lines at day 7, −/+ DOX treatment to overexpress ZIC1 starting at day 3 (n = 2 and 3 independent experiments) or day 4 (n = 3 independent experiments). All data shown as mean ± SEM, ^∗p < 0.05. See also Figure S3.

Figure 4

Expression of Truncated ASXL1 Impairs NC Cell Emigration and ZIC1 Expression (A) Detection of ASXL1 by western blotting in samples of day 7 NC cultures (monoclonal antibody as in Figures 1E and 1F). (B) Percentage of attached neurospheres with emigrating cells derived at day 7 from DOX-treated ASXL1^+/+ hESCs and PB-ASXL1^PSC hESCs, overexpressing truncated ASXL1, relative to untreated samples (mean ± SEM, n = 3 independent experiments, ^∗p < 0.05). (C) Representative fluorescent images of chicken embryos 48 h after electroporation with plasmids expressing GFP or GFP coupled to truncated chicken or human ASXL1^PSC (n = 10 embryos each); the red rectangle indicates displayed head/hindbrain region. Scale bars, 200 μm. (D) Expression pattern of ZIC1 analyzed by in situ hybridization in neural tube sections of chicken embryos (HH15) electroporated with plasmids encoding for truncated (ASXL^PSC) or full-length chicken ASXL1 coupled to GFP. I, injected side; U, control side. n = 3 sections per 2 embryos. Scale bar, 50 μm. (E) Representative bright-field image of chicken embryo electroporated with truncated chicken ASXL1 transcript as in (C), showing craniofacial malformations (arrow, missing periocular tissue) on the injected side in comparison with the uninjected control (CTRL) side. n = 6 embryos. Scale bars, 200 μm. See also Figure S4.