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• W2936707944 abstract "•Generation of human pluripotent stem cell models for Bohring-Opitz syndrome•ASXL1 mutations perturb neural crest (NC) emigration in vitro and in vivo•ASXL1 mutations correlate with impaired activation of ZIC1•Expression of truncated ASXL1 variants mirrors NC perturbation in vitro and in vivo 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. 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. The neural crest (NC) is an embryonic progenitor population that gives rise to multiple derivatives, including craniofacial cartilage and bones and peripheral neurons, many of which are associated with birth defects such as craniofacial and cardiac malformations (Etchevers et al., 2006Etchevers H.C. Amiel J. Lyonnet S. Molecular bases of human neurocristopathies.Adv. Exp. Med. Biol. 2006; 589: 213-234Crossref PubMed Scopus (73) Google Scholar, Mayor and Theveneau, 2013Mayor R. Theveneau E. The neural crest.Development. 2013; 140: 2247-2251Crossref PubMed Scopus (204) Google Scholar, Snider and Mishina, 2014Snider T.N. Mishina Y. Cranial neural crest cell contribution to craniofacial formation, pathology, and future directions in tissue engineering.Birth Defects Res. C Embryo Today. 2014; 102: 324-332Crossref PubMed Scopus (26) Google Scholar). Animal studies have shown that initiation of NC development at the neural plate border, as well as subsequent delamination, migration, and terminal differentiation of the NC progenitors, are orchestrated by specific gene regulatory networks (GRNs) (Simoes-Costa and Bronner, 2015Simoes-Costa M. Bronner M.E. Establishing neural crest identity: a gene regulatory recipe.Development. 2015; 142: 242-257Crossref PubMed Scopus (361) Google Scholar). Advancing the understanding of these GRNs in human NC development necessitates modeling by pluripotent stem cells, in particular for investigating the epigenetic mechanisms that regulate them (Bajpai et al., 2010Bajpai R. Chen D.A. Rada-Iglesias A. Zhang J. Xiong Y. Helms J. Chang C.P. Zhao Y. Swigut T. Wysocka J. CHD7 cooperates with PBAF to control multipotent neural crest formation.Nature. 2010; 463: 958-962Crossref PubMed Scopus (426) Google Scholar). Nevertheless, the involvement of mutations in histone-modifying enzymes in pathological NC development has not yet been studied. Bohring-Opitz syndrome (BOS) is a severe congenital disorder associated with de novo mutations in the Additional sex combs-like 1 (ASXL1) gene, characterized by symptoms that include developmental delay and musculoskeletal and neurological features (Hastings et al., 2011Hastings R. Cobben J.M. Gillessen-Kaesbach G. Goodship J. Hove H. Kjaergaard S. Kemp H. Kingston H. Lunt P. Mansour S. et al.Bohring-Opitz (Oberklaid-Danks) syndrome: clinical study, review of the literature, and discussion of possible pathogenesis.Eur. J. Hum. Genet. 2011; 19: 513-519Crossref PubMed Scopus (34) Google Scholar). ASXL1 is a co-factor of the Polycomb repressive complex 2 (PRC2), and loss of function (Abdel-Wahab et al., 2012Abdel-Wahab O. Adli M. LaFave L.M. Gao J. Hricik T. Shih A.H. Pandey S. Patel J.P. Chung Y.R. Koche R. et al.ASXL1 mutations promote myeloid transformation through loss of PRC2-mediated gene repression.Cancer Cell. 2012; 22: 180-193Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar) and expression of dominantly acting truncated ASXL1 variants (Balasubramani et al., 2015Balasubramani A. Larjo A. Bassein J.A. Chang X. Hastie R.B. Togher S.M. Lahdesmaki H. Rao A. Cancer-associated ASXL1 mutations may act as gain-of-function mutations of the ASXL1-BAP1 complex.Nat. Commun. 2015; 6: 7307Crossref PubMed Scopus (130) Google Scholar, Guo et al., 2018Guo Y. Yang H. Chen S. Zhang P. Li R. Nimer S.D. Harbour J.W. Xu M.J. Yang F.C. Reduced BAP1 activity prevents ASXL1 truncation-driven myeloid malignancy in vivo.Leukemia. 2018; 32: 1834-1837Crossref PubMed Scopus (16) Google Scholar) have been associated with transcriptional misregulations in hematological disorders. Whether and how these mechanisms relate to the molecular mechanism of BOS is unknown. Here, we reasoned that craniofacial malformations such as palate anomalies indicate a potential perturbation of NC development in BOS (Hastings et al., 2011Hastings R. Cobben J.M. Gillessen-Kaesbach G. Goodship J. Hove H. Kjaergaard S. Kemp H. Kingston H. Lunt P. Mansour S. et al.Bohring-Opitz (Oberklaid-Danks) syndrome: clinical study, review of the literature, and discussion of possible pathogenesis.Eur. J. Hum. Genet. 2011; 19: 513-519Crossref PubMed Scopus (34) Google Scholar). By analyzing a comprehensive set of genetically edited human embryonic stem cell (hESC) and patient induced pluripotent stem cell (iPSC) lines that we differentiated into NC progenitors, we identified transcriptional perturbations associated with defects in NC development, which we propose provide a putative mechanism for the craniofacial and plausibly other symptoms in BOS patients. We obtained dermal fibroblasts from two BOS patients who carry monoallelic premature stop codons (PSCs) in the terminal exon of ASXL1 (Magini et al., 2012Magini P. Della Monica M. Uzielli M.L. Mongelli P. Scarselli G. Gambineri E. Scarano G. Seri M. Two novel patients with Bohring-Opitz syndrome caused by de novo ASXL1 mutations.Am. J. Med. Genet. A. 2012; 158A: 917-921Crossref PubMed Scopus (33) Google Scholar). Patient fibroblasts were reprogrammed using two techniques, by episomal plasmids and modified mRNAs, resulting in the derivation of four BOS-iPSC lines (Figure 1A, Table S1, Figure S1A). In addition, we used CRISPR/Cas9 to excise a 500 bp region from the terminal exon of ASXL1 in hESCs, generating clones that harbor heterozygous and homozygous BOS patient-like PSC mutations (ASXL1PSC/+ and ASXL1PSC/PSC; Figure 1A, Table S1, Figures S1B and S1C). BOS-iPSC and edited hESC lines expressed canonical pluripotency markers and displayed colony morphology comparable with control lines, which were the parental ASXL1+/+ hESC line and two control iPSC lines, respectively (Figure S1A and S1C–S1G, Table S1). Plausible molecular mechanisms of BOS include ASXL1 haploinsufficiency and/or expression of truncated ASXL1 (Figure 1B). Testing the level of ASXL1 transcripts in undifferentiated cells did not reveal a difference between BOS-iPSC, ASXL1PSC/+, and ASXL1PSC/PSC clones and the respective control lines (Figure 1C). We confirmed the presence of mutant ASXL1 transcripts in the genetically edited hESC clones, and in both BOS-iPSC lines #1-0 and #2-0 by Sanger sequencing (Figure 1D). Furthermore, we detected comparable levels of the normal and mutant transcripts using a primer set specifically targeting the 5-bp deletion in BOS-iPSC lines #1-0/#1-1 (Figure S1H). To further investigate the expression of truncated ASXL1 variants, we raised a monoclonal antibody targeting the N terminus of ASXL1, which by western blotting detected a truncated ASXL1 variant that was overexpressed in undifferentiated hESCs (PB-ASXL1PSC; Table S1, Figure 1E). This antibody also detected putative truncated protein isoforms in BOS-iPSC lines #1 and #2, and ASXL1PSC/PSC hESC clones (Figures 1F and S1I), albeit at varying levels (Figure S1J). Collectively, this indicates that expression of truncated ASXL1 variants is a possible mode of misregulation in BOS. Craniofacial symptoms that are common in BOS patients prompted us to investigate a possible connection to the NC lineage in the hESC and iPSC models. We utilized a differentiation protocol that generated delaminating NC-like cells (Bajpai et al., 2010Bajpai R. Chen D.A. Rada-Iglesias A. Zhang J. Xiong Y. Helms J. Chang C.P. Zhao Y. Swigut T. Wysocka J. CHD7 cooperates with PBAF to control multipotent neural crest formation.Nature. 2010; 463: 958-962Crossref PubMed Scopus (426) Google Scholar) from neuroepithelial spheres (neurospheres; Figure 2A), which expressed the characteristic NC markers p75NTR, SOX9, SOX10, TFAP2A, PAX3, and SNAI2 (Lee et al., 2007Lee G. Kim H. Elkabetz Y. Al Shamy G. Panagiotakos G. Barberi T. Tabar V. Studer L. Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells.Nat. Biotechnol. 2007; 25: 1468-1475Crossref PubMed Scopus (424) Google Scholar, Simoes-Costa and Bronner, 2015Simoes-Costa M. Bronner M.E. Establishing neural crest identity: a gene regulatory recipe.Development. 2015; 142: 242-257Crossref PubMed Scopus (361) Google Scholar) and the proliferation marker KI67 (Figures S2A and S2B). Moreover, in accordance with the expected potency of NC progenitors, they readily gave rise to putative mesenchymal stem cells (MSCs) (Noden and Trainor, 2005Noden D.M. Trainor P.A. Relations and interactions between cranial mesoderm and neural crest populations.J. Anat. 2005; 207: 575-601Crossref PubMed Scopus (320) Google Scholar) that exhibited spindle-shaped morphology, expression of consensus MSC surface markers CD73, CD90, and CD105 (Ramos et al., 2016Ramos T.L. Sanchez-Abarca L.I. Muntion S. Preciado S. Puig N. Lopez-Ruano G. Hernandez-Hernandez A. Redondo A. Ortega R. Rodriguez C. et al.MSC surface markers (CD44, CD73, and CD90) can identify human MSC-derived extracellular vesicles by conventional flow cytometry.Cell Commun. Signal. 2016; 14: 2Crossref PubMed Scopus (169) Google Scholar), and terminally differentiated into osteoblasts and adipocytes (Figures S2C–S2E). We noted upregulation of ASXL1 and ASXL3, but not of ASXL2, in NC progenitors (Figure S2F), indicating specific roles of the two genes during NC differentiation, which was further supported by co-expression of ASXL1 and the NC regulator TFAP2A (Figure S2G). Importantly, neurospheres derived from BOS-iPSC lines with heterozygous ASXL1 mutations displayed reduced attachment and diminished emigration of NC cells in comparison with control iPSC-derived neurospheres (Figure 2B, left panel). In the genetically edited hESC lines, we observed a similar reduction in neurosphere attachment and NC emigration, an effect that was statistically significant only in homozygous ASXL1PSC/PSC clones (Figure 2B, right panel), with the presence of dead, floating cells surrounding the neurospheres (Figures 2C and S2H). ASXL1 mutations did not negatively affect the propensity to form neurospheres; conversely, an increase in the total number of neurospheres was observed in lines harboring mutant ASXL1 (Figure S2I). Thus, ASXL1 mutations perturbed the generation and/or delamination of the NC in vitro, potentially via defective induction or specification already at the neurosphere stage. To further test the emigration of NC cells in vivo, we performed orthotopic xenotransplantation of neurospheres obtained from eGFP-labeled ASXL1PSC/PSC and ASXL+/+ hESC lines (Table S1) into Hamburger-Hamilton (HH) stage 10 chicken embryos (Figure 2D). Neurospheres derived from ASXL1PSC/PSC hESC clones exhibited a significant reduction in the number of emigrating cells compared with neurospheres derived from the parental ASXL1+/+ hESC line (Figures 2D and 2E), an effect that was not due to size differences between control and mutant neurospheres (Figure S2J). Cells that emigrated from the neurospheres of both lines transversed a similar distance (Figure S2K). Collectively, these results indicate a connection between ASXL1, NC development, and BOS. To delineate mechanisms underlying the paucity and migration defect of the NC progenitors, we conducted global RNA sequencing. This revealed mutually exclusive clustering of samples derived from NC progenitors harboring the ASXL1PSC/PSC genotype and the parental control cells (Figure S3A). Strikingly, the neural plate border transcription factors ZIC1 and ZIC4 (Merzdorf, 2007Merzdorf C.S. Emerging roles for zic genes in early development.Dev. Dyn. 2007; 236: 922-940Crossref PubMed Scopus (119) Google Scholar), which are an immediately adjacent gene pair, were among the most negatively regulated transcripts in the ASXL1PSC/PSC NC progenitors (Figure 3A). This substantiated the link to BOS and related syndromes, because heterozygous deletion of the ZIC1/ZIC4 locus is associated with Dandy-Walker malformation (Grinberg et al., 2004Grinberg I. Northrup H. Ardinger H. Prasad C. Dobyns W.B. Millen K.J. Heterozygous deletion of the linked genes ZIC1 and ZIC4 is involved in Dandy-Walker malformation.Nat. Genet. 2004; 36: 1053-1055Crossref PubMed Scopus (181) Google Scholar), a defect in cerebellar development that is observed in BOS patients (Hastings et al., 2011Hastings R. Cobben J.M. Gillessen-Kaesbach G. Goodship J. Hove H. Kjaergaard S. Kemp H. Kingston H. Lunt P. Mansour S. et al.Bohring-Opitz (Oberklaid-Danks) syndrome: clinical study, review of the literature, and discussion of possible pathogenesis.Eur. J. Hum. Genet. 2011; 19: 513-519Crossref PubMed Scopus (34) Google Scholar). Moreover, negatively regulated genes in the dataset were significantly associated with several disorders and malformations found in BOS, including agenesis of the corpus callosum, craniofacial dysmorphisms, musculoskeletal abnormalities, and seizures (Figure 3B). ZIC1, which was 50-fold reduced in ASXL1PSC/PSC NC cultures, is at the apex of the GRN controlling NC induction (Simoes-Costa and Bronner, 2015Simoes-Costa M. Bronner M.E. Establishing neural crest identity: a gene regulatory recipe.Development. 2015; 142: 242-257Crossref PubMed Scopus (361) Google Scholar). Accordingly, transcription factors that promote further specification, delamination, and migration of the NC were also negatively regulated in the ASXL1PSC/PSC NC cells, including PAX3, GBX2, MSX1, NR2F1/2, ZEB2, COL2A1, TWIST1, SOX5, Cadherins, and Ephrins and their cognate receptors (Rada-Iglesias et al., 2012Rada-Iglesias A. Bajpai R. Prescott S. Brugmann S.A. Swigut T. Wysocka J. Epigenomic annotation of enhancers predicts transcriptional regulators of human neural crest.Cell Stem Cell. 2012; 11: 633-648Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, Simoes-Costa and Bronner, 2015Simoes-Costa M. Bronner M.E. Establishing neural crest identity: a gene regulatory recipe.Development. 2015; 142: 242-257Crossref PubMed Scopus (361) Google Scholar) (Figures 3C, S3B, and S3C, Table S2). qPCR, performed to confirm the transcriptional changes for a subset of NC genes, demonstrated significant downregulation in the ASXL1PSC/PSC clones and mild reduction in the patient lines (Figures 3D and 3E), and ZIC1 protein levels were significantly lower in NC cultures derived from one BOS-iPSC line (Figures 3F and S3D). To further analyze the connection of the ASXL1PSC/+ ESC line to NC defects, we performed 3′ mRNA sequencing of NC cultures derived from the heterozygous ASXLPSC/+ clones and control hESCs. This revealed mutually exclusive clustering of the two genotypes and a significant 28-fold downregulation of ZIC1 in ASXLPSC/+ NC progenitors (padj < 0.05; Figures S3E–S3G). Collectively, this supports a link between BOS-associated mutations in ASXL1 and ZIC1 misregulation. To investigate whether reduced ZIC1 levels could indeed underlie the observed NC differentiation phenotypes, we stably integrated an inducible ZIC1 transgene in ASXL1+/+ and ASXL1PSC/PSC hESC clones (Figure 3G, Table S1). Strikingly, overexpression of ZIC1 during NC differentiation restored the attachment and delamination phenotype of NC-like cells from ASXL1PSC/PSC neurospheres (Figure 3H). These results collectively indicate that impairment of ZIC1 activation contributes to the observed differentiation defects when ASXL1 is mutated. We next investigated how regulation of ASXL1 could be implicated in the differentiation outcomes displayed by the BOS models. We noted decreased expression of ASXL1 in NC progenitors derived from ASXL1PSC/+ and ASXL1PSC/PSC hESC, and a similar effect for the BOS-iPSC lines, which was however not deemed statistically significant (Figure S4A). Moreover, mutant and wild-type ASXL1 transcripts were found in NC cultures derived from BOS #1 and #2 lines (Figure S4B), and both alleles were expressed at comparable levels in BOS-iPSC #1 lines (Figure S4C). This argues against degradation of the mutant mRNA. We found that overexpression of wild-type ASXL1 by a stably integrated PiggyBac vector (Table S1) could not rescue the attachment defect of neurospheres derived from ASXL1PSC/PSC hESCs, nor did it rescue expression of key NC genes (Figures S4D and S4F). These findings suggest a dominant role of putative truncated ASXL1 variants over the wild-type form. Western blot analysis of early NC cells from all BOS models, using the monoclonal antibody raised against the N-terminal domain of ASXL1, revealed barely detectable levels of a putative truncated variant at 100 kDa and enhanced levels of different variants at 130 and 160 kDa (Figure 4A). Collectively, these results raise the possibility that truncated ASXL1 variants, reduction in ASXL1 levels, and potentially also misexpression of additional ASXL1 variants (Figures S1I, S1J, and 4A) contributed to the observed NC defects. To substantiate one of these possibilities, we expressed in hESCs a truncated ASXL1 isoform, which was similar to the variant detected in BOS patient 2 (Table S1). Such ectopic expression led to a nearly complete failure of neurospheres to attach and produce migratory NC-like cells (Figure 4B). Moreover, truncated human as well as chicken ASXL1 that were injected into the anterior neural region of chicken embryos inhibited the delamination of the electroporated GFP-labeled cells, an effect that was not seen in embryos injected with control plasmid encoding only eGFP (Figure 4C). Strikingly, ZIC1 expression was reduced in neural tube cells expressing truncated chicken ASXL1 but not in embryos electroporated with full-length chicken ASXL1 (Figures 4D and S4G). Under the same conditions, expression of TFAP2A was unaltered (Figure S4G), indicating a specific perturbation of ZIC1 expression that was similar to the case of ASXL1PSC/PSC NC progenitors in vitro (Figure S3B). Finally, we analyzed electroporated embryos at later developmental stages, around HH22–25, to assess craniofacial phenotypes caused by expression of truncated ASXL1. This revealed decreased eye size and malformation of periocular tissue on the injected side compared with the uninjected side (Figure 4E). We thus concluded that expression of truncated ASXL1 in NC progenitors could lead to BOS-like phenotypes in vivo. Collectively, in this study we created a human pluripotent stem cell toolset for investigating pathological ASXL1 mutations present in, or resembling genotypes of, BOS patients. These models enabled us to discover the impaired generation of NC cells, which might be partially caused by a defect in early neuroectoderm specification, and our results support an association with reduced induction of ZIC1. However, the question of whether the truncated ASXL1 isoforms by themselves, haploinsufficiency, other isoforms, or combinations of these mechanisms underlie BOS pathogenesis warrants additional studies. A similar question exists with respect to truncated endogenous ASXL1 variants that are associated with leukemia (Abdel-Wahab et al., 2012Abdel-Wahab O. Adli M. LaFave L.M. Gao J. Hricik T. Shih A.H. Pandey S. Patel J.P. Chung Y.R. Koche R. et al.ASXL1 mutations promote myeloid transformation through loss of PRC2-mediated gene repression.Cancer Cell. 2012; 22: 180-193Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, Balasubramani et al., 2015Balasubramani A. Larjo A. Bassein J.A. Chang X. Hastie R.B. Togher S.M. Lahdesmaki H. Rao A. Cancer-associated ASXL1 mutations may act as gain-of-function mutations of the ASXL1-BAP1 complex.Nat. Commun. 2015; 6: 7307Crossref PubMed Scopus (130) Google Scholar, Inoue et al., 2016Inoue D. Matsumoto M. Nagase R. Saika M. Fujino T. Nakayama K.I. Kitamura T. Truncation mutants of ASXL1 observed in myeloid malignancies are expressed at detectable protein levels.Exp. Hematol. 2016; 44: 172-176.e1Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Thereby, our findings and models are likely pertinent to hematological malignancies. We moreover linked the truncated isoform to craniofacial dysmorphisms, which have strong association with BOS, and therefore propose ASXL1-mediated perturbation of the NC as a potential cause of characteristic symptoms in BOS. A further medical relevance is apparent from the symptomatic similarity of BOS to Bainbridge-Roper syndrome (BRS), a congenital disorder that is associated with ASXL3 mutations (Bainbridge et al., 2013Bainbridge M.N. Hu H. Muzny D.M. Musante L. Lupski J.R. Graham B.H. Chen W. Gripp K.W. Jenny K. Wienker T.F. et al.De novo truncating mutations in ASXL3 are associated with a novel clinical phenotype with similarities to Bohring-Opitz syndrome.Genome Med. 2013; 5: 11Crossref PubMed Scopus (113) Google Scholar), which raises the possibility that BRS is caused by similar mechanisms. The results may also explain the occurrence of Dandy-Walker malformation in BOS patients that could take place due to ZIC1/ZIC4 repression (Grinberg et al., 2004Grinberg I. Northrup H. Ardinger H. Prasad C. Dobyns W.B. Millen K.J. Heterozygous deletion of the linked genes ZIC1 and ZIC4 is involved in Dandy-Walker malformation.Nat. Genet. 2004; 36: 1053-1055Crossref PubMed Scopus (181) Google Scholar). Finally, the speech and language learning disabilities in BOS (Hastings et al., 2011Hastings R. Cobben J.M. Gillessen-Kaesbach G. Goodship J. Hove H. Kjaergaard S. Kemp H. Kingston H. Lunt P. Mansour S. et al.Bohring-Opitz (Oberklaid-Danks) syndrome: clinical study, review of the literature, and discussion of possible pathogenesis.Eur. J. Hum. Genet. 2011; 19: 513-519Crossref PubMed Scopus (34) Google Scholar) may be linked to the reduced FOXP2 activation (Figure 3A), as this transcription factor has a primary role in speech and language development (Enard et al., 2002Enard W. Przeworski M. Fisher S.E. Lai C.S. Wiebe V. Kitano T. Monaco A.P. Paabo S. Molecular evolution of FOXP2, a gene involved in speech and language.Nature. 2002; 418: 869-872Crossref PubMed Scopus (1033) Google Scholar). Overall, the established models could help elucidate ASXL1-related pathological mechanisms in additional tissues, including the nervous system and the hematopoietic lineage. Expression and purification of an N-terminal fragment of ASXL1 (amino acids 1–618), followed by immunization of Lou/c rats was performed as previously described (Feederle et al., 2016Feederle R. Gerber J.K. Middleton A. Northrup E. Kist R. Kremmer E. Peters H. Generation of Pax1/PAX1-specific monoclonal antibodies. Monoclonal antibodies in immunodiagnosis and immunotherapy.Monoclon. Antib. Immunodiagn. Immunother. 2016; 35: 259-262Crossref PubMed Scopus (7) Google Scholar, Studier, 2005Studier F.W. Protein production by auto-induction in high density shaking cultures.Protein Expr. Purif. 2005; 41: 207-234Crossref PubMed Scopus (4106) Google Scholar). Fibroblasts derived from skin biopsies of healthy donors and BOS patients under oversight by local institutional review boards (Magini et al., 2012Magini P. Della Monica M. Uzielli M.L. Mongelli P. Scarselli G. Gambineri E. Scarano G. Seri M. Two novel patients with Bohring-Opitz syndrome caused by de novo ASXL1 mutations.Am. J. Med. Genet. A. 2012; 158A: 917-921Crossref PubMed Scopus (33) Google Scholar) were reprogrammed by modified mRNA and episomal plasmids (Diecke et al., 2015Diecke S. Lu J. Lee J. Termglinchan V. Kooreman N.G. Burridge P.W. Ebert A.D. Churko J.M. Sharma A. Kay M.A. et al.Novel codon-optimized mini-intronic plasmid for efficient, inexpensive, and xeno-free induction of pluripotency.Sci. Rep. 2015; 5: 8081Crossref PubMed Scopus (45) Google Scholar, Kunze et al., 2018Kunze C. Borner K. Kienle E. Orschmann T. Rusha E. Schneider M. Radivojkov-Blagojevic M. Drukker M. Desbordes S. Grimm D. et al.Synthetic AAV/CRISPR vectors for blocking HIV-1 expression in persistently infected astrocytes.Glia. 2018; 66: 413-427Crossref PubMed Scopus (45) Google Scholar), and 500 bp deletions in the ASXL1 gene were induced in iCas9 HUES9 hESC (ASXL1+/+ control line) (Gonzalez et al., 2014Gonzalez F. Zhu Z. Shi Z.D. Lelli K. Verma N. Li Q.V. Huangfu D. An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells.Cell Stem Cell. 2014; 15: 215-226Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). ASXL1PSC/PSC and ASXL1+/+ hESC lines were stably integrated with PiggyBac vectors harboring either the truncated ASXL1 cDNA sequence (N-terminal 2,892 bp; PB-ASXL1PSC), the full-length human ASXL1 cDNA (4,656 bp; PB-ASXL1), the ZIC1 cDNA sequence (amplified from ZIC1 human cDNA clone, Biocat; PB-ZIC1), or an expression cassette for continuous eGFP expression. Neural crest differentiation was performed as described (Bajpai et al., 2010Bajpai R. Chen D.A. Rada-Iglesias A. Zhang J. Xiong Y. Helms J. Chang C.P. Zhao Y. Swigut T. Wysocka J. CHD7 cooperates with PBAF to control multipotent neural crest formation.Nature. 2010; 463: 958-962Crossref PubMed Scopus (426) Google Scholar), and the number of attached neurospheres was quantified at day 7 of the protocol. According to German animal care guidelines, no IACUC (Institutional Animal Care and Use Committee) approval was necessary to perform chicken embryo experiments. Neurospheres obtained at day 5 of NC differentiation were inserted into the developing anterior neural region of chicken embryos (HH10; Hamburger and Hamilton, 1992Hamburger V. Hamilton H.L. A series of normal stages in the development of the chick embryo. 1951.Dev. Dyn. 1992; 195: 231-272Crossref PubMed Scopus (1441) Google Scholar) and operated embryos were isolated at HH22 and analyzed under a fluorescence stereo microscope. To overexpress transcripts in chicken embryos, the truncated chicken (Gallus gallus) ASXL1 cDNA sequence (Gg-ASXL1PSC; N-terminal 2,445 bp), the truncated human ASXL1 cDNA sequence (hASXL1PSC; N-terminal 2,892 bp), or the full-length chicken ASXL1 cDNA sequence (Gg-ASXL1; 4,617 bp) were cloned into expression plasmids and electroporated into the developing brain and neural tube at HH9–10 as described (Rehimi et al., 2016Rehimi R. Nikolic M. Cruz-Molina S. Tebartz C. Frommolt P. Mahabir E. Clement-Ziza M. Rada-Iglesias A. Epigenomics-based identification of major cell identity regulators within heterogeneous cell populations.Cell Rep. 2016; 17: 3062-3076Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Manipulated chicken embryos were imaged at HH19 and HH24–25 under a fluorescence stereo microscope. In situ hybridization experiments in electroporated chicken embryos were performed as described (Rehimi et al., 2016Rehimi R. Nikolic M. Cruz-Molina S. Tebartz C. Frommolt P. Mahabir E. Clement-Ziza M. Rada-Iglesias A. Epigenomics-based identification of major cell identity regulators within heterogeneous cell populations.Cell Rep. 2016; 17: 3062-3076Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Total RNA was isolated from day 7 NC progenitor cultures and libraries were prepared using the TruSeq Stranded Total RNA LT Library Prep Kit (Illumina) followed by single-end sequencing. Differential gene expression analysis was performed with the DESeq2 package (Love et al., 2014Love M.I. Huber W. Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol. 2014; 15: 550Crossref PubMed Scopus (32318) Google Scholar). Values are expressed as means ± standard error of the mean; pairwise comparison was performed using Welch's t test or Wilcoxon’s rank-sum test. F.M. and M.D. designed the study. F.M., E.R., and L.M. performed functional experiments. E.R. and A.P. generated iPSC lines. M.M. performed library preparation and analyzed 3′ RNA sequencing experiments. R.R. and A.R.-I. contributed to the chicken embryo experiments. E.K., R.F., A.F., and A.G. produced monoclonal antibodies. V.R. performed MSC analysis. F.M. and M.D. wrote the manuscript with input from the other authors. We thank the patients' families and following scientists for generously providing fibroblasts: Pamela Magini (S. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy), Matteo Della Monica (Meyer Children's Hospital, University of Florence, Florence, Italy), Gioacchino Scarano (Medical Genetics Unit, A.O.R.N “G. Rummo”, Benevento, Italy), and Prof. Maria Luisa Giovannucci Uzielli (Genetics and Molecular Medicine, University of Florence, Florence, Italy). We would also like to thank Meino Rohlfs and Jens Hinke (Klinikum LMU, Munich) and Christine Wurmser (TU Munich) for providing help with RNA sequencing, Lukas Simon (Helmholtz Zentrum München, Neuherberg) for support with bioinformatics data analysis, and Dmitry Shaposhnikov (Helmholtz Zentrum München) for providing plasmids. Gene expression data from the RNA sequencing experiments have been deposited in NCBI's Gene Expression Omnibus (GEO) under accession numbers GEO: GSE120200 and GSE127269. Download .pdf (26.12 MB) Help with pdf files Document S1. Supplemental Experimental Procedures, Figures S1–S4, and Tables S1, S3, and S4 Download .xlsx (.14 MB) Help with xlsx files Table S2. Downregulated Genes in ASXL1 Mutant NC Progenitor Cultures, Related to Figure 3" @default.
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