Stem Cell Reports. 2019 May 14;12(5):861-868. doi: 10.1016/j.stemcr.2019.03.006.

Pathological ASXL1 Mutations and Protein Variants Impair Neural Crest Development

Matheus F1, Rusha E2, Rehimi R3, Molitor L1, Pertek A2, Modic M4, Feederle R5, Flatley A5, Kremmer E6, Geerlof A7, Rishko V1, Rada-Iglesias A3, Drukker M8.
1 Institute for Stem Cell Research, Helmholtz Zentrum München GmbH, 85764 Neuherberg, Germany.
2 Institute for Stem Cell Research, iPSC Core Facility, Helmholtz Zentrum München GmbH, 85764 Neuherberg, Germany.
3 Center for Molecular Medicine Cologne (CMMC), 50931 Köln, Germany.
4 The Francis Crick Institute, London NW1 1AT, UK; Department for Neuromuscular Diseases, UCL Queen Square Institute of Neurology, London WC1N 3BG, UK.
5 Institute for Diabetes and Obesity, Monoclonal Antibody Core Facility, Helmholtz Zentrum München GmbH, 85764 Neuherberg, Germany.
6 Institute of Molecular Immunology, Helmholtz Zentrum München GmbH, 85764 Neuherberg, Germany.
7 Institute of Structural Biology, Protein Expression and Purification Facility, Helmholtz Zentrum München GmbH, 85764 Neuherberg, Germany.
8 Institute for Stem Cell Research, Helmholtz Zentrum München GmbH, 85764 Neuherberg, Germany. Electronic address:


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

PMID: 31006630



Congenital disorders are major causes of severe chronic disabilities and infant mortality. It is estimated that over 500 mendelian congenital disorders involve perturbation of the neural crest (NC) [1]. The NC is a multipotent, transient embryonic cell population that first forms at the neural plate border, and later migrates throughout the embryo, contributing to various organs and differentiating into a plethora of cell types, including bones of the skull, melanocytes, and the enteric nervous system [2]. This enormous developmental potential demands tight control of NC formation, proliferation, migration and differentiation to specific cell types. Epigenetic regulation of human NC development and the role of histone-modifying enzymes in NC-related disorders are however poorly understood.


In our work, we link the congenital disorder Bohring-Opitz syndrome (BOS) to perturbed development of the human NC. BOS is characterized by musculoskeletal features, severe feeding difficulties, craniofacial dysmorphisms, severe developmental delay, intellectual disabilities, and high risk of death in early infancy [3, 4]. The congenital disorder has recently been associated with mutations in the chromatin regulator Additional sex combs-like 1 (ASXL1) [5]. ASXL1 acts as a regulatory scaffold that assembles transcription factors and repressive or activating histone modifiers, including the Polycomb repressive complex 2 complex, to promote global or local deposition of chromatin marks.  Strikingly, similar to the case of ASXL1-associated myeloid cancers, all the ASXL1 mutations reported in BOS patients lie in the penultimate or last exon of the gene, hinting towards similar molecular mechanisms. However, it has not been clear whether BOS-causing mutations result in ASXL1 loss of function or a dominant functionality of the truncated protein during embryonic development. The pending clarification of a molecular etiology is of special importance, as the other ASXL family members are likewise implicated in congenital malformations.


To address this question, we developed a comprehensive set of pluripotent stem cell lines consisting of patient-derived induced pluripotent stem (iPS) cells and genetically edited human embryonic stem (ES) cells, which we used to study BOS in vitro. In these BOS cell lines, we found expression of a putative truncated ASXL1 protein corresponding to aberrant ASXL1 variants reported in myeloid cancer cell lines [6].


The broad range of symptoms in BOS, and also specific features like the craniofacial dysmorphisms, prompted us to analyze the involvement of the NC. This hypothesis is compliant with the expression patterns of Asxl1 in NC-associated tissues of the mouse fetus [7]. By differentiating the BOS stem cell lines in vitro, we discovered reduced generation and migration of NC progenitors, which we validated by engrafting human NC progenitors in chicken embryos (Fig. 1).


To better understand the molecular mechanisms underlying these findings, we analyzed the transcriptomes of BOS NC cultures by next generation sequencing, which led us to identify reduced expression of the NC regulatory genetic network. In particular, decreased activation of the NC transcription factor ZIC1 seemed to be a crucial bottleneck impeding NC differentiation in BOS lines. Evidence that ectopic expression of ZIC1 partially restored the developmental defect in these models supported this mechanism. Remarkably, our observations resonate with the known role of Zic1 being one master transcription factor at the apex of the NC regulatory network [8].


To furthermore investigate whether truncated ASXL1 could be underlying the observed developmental and transcriptional effects, we expressed a truncated ASXL1 gene in the developing neural crest in ovo. This strongly inhibited the emigration of the targeted cells and led to craniofacial abnormalities in the electroporated embryos. Together with further transcriptional analyses in our cell systems, we conclude that possibly a combination of truncated and alternative splice variants of ASXL1 and reduced expression of the normal ASXL1 underlie the NC-related symptoms in our study. Further studies should shed light on the stability and expression of different ASXL1 variants in BOS, and their activity in recruiting chromatin modifiers like BAP1 [9].


In recent years, a growing list of human congenital symptoms has been linked to mutations in the three ASXL genes. We outline with our study a first mechanistic understanding of BOS and of the pathological roles of ASXL1 mutations, linking them to NC development, and providing hints towards additional unexplained malformations. Our findings are medically relevant because as we show, the transcripts altered in BOS NC progenitor cultures are associated with several common developmental syndromes including craniofacial abnormalities and neural tube defects. Moreover, co-regulation of ASXL1’s paralog ASXL3 suggests that the dysmorphisms associated with mutations of ASXL3 in Bainbridge Ropers syndrome (BRS) [10] partly originate in NC progenitors. The development of BRS stem cell models should elucidate this point further.


Our findings can thus be an important foundation for future research on therapeutic intervention in BOS and related disorders. The gastrointestinal problems observed in BOS patients could be one important starting point in this context, as they might hint towards a dysfunctional enteric nervous system, which in turn conceivably results from perturbed NC development in these patients.



Figure 1. Representative fluorescent images of neurospheres that were differentiated from GFP-labeled hESC lines harbouring wildtype (GFP-ASXL1+/+) or genetically edited ASXL1 alleles (GFP-ASXL1PSC/PSC) and then transplantated into the hindbrain region of chicken embryos. Scale bar: 200 µm; pictures taken 48 h after transplantation.



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