Functional perturbation and action mechanism studies have shown that the transcription factor Zeb2 controls cell fate decisions, differentiation, and/or maturation in multiple cell lineages in embryos and after birth. In cultured embryonic stem cells (ESCs), Zeb2’s mRNA/protein upregulation is necessary for the exit from primed pluripotency and for entering general and neural differentiation. We edited mouse ESCs to produce Flag-V5 epitope-tagged Zeb2 protein from one endogenous allele. Using chromatin immunoprecipitation coupled with sequencing (ChIP-seq), we mapped 2432 DNA-binding sites for this tagged Zeb2 in ESC-derived neuroprogenitor cells (NPCs). A new, major binding site maps promoter-proximal to Zeb2 itself. The homozygous deletion of this site demonstrates that autoregulation of Zeb2 is necessary to elicit the appropriate Zeb2-dependent effects in ESC-to-NPC differentiation. We have also cross-referenced all the mapped Zeb2 binding sites with previously obtained transcriptome data from Zeb2 perturbations in ESC-derived NPCs, GABAergic interneurons from the ventral forebrain of mouse embryos, and stem/progenitor cells from the post-natal ventricular-subventricular zone (V-SVZ) in mouse forebrain, respectively. Despite the different characteristics of each of these neurogenic systems, we found interesting target gene overlaps. In addition, our study also contributes to explaining developmental disorders, including Mowat-Wilson syndrome caused by ZEB2 deficiency, and also other monogenic syndromes.

Neurodevelopmental disorders (NDDs) are a heterogeneous group of impairments that affect the development of the central nervous system leading to abnormal brain function. NDDs affect a great percentage of the population worldwide, imposing a high societal and economic burden and thus, interest in this field has widely grown in recent years. Nevertheless, the complexity of human brain development and function as well as the limitations regarding human tissue usage make their modeling challenging. Animal models play a central role in the investigation of the implicated molecular and cellular mechanisms, however many of them display key differences regarding human phenotype and in many cases, they partially or completely fail to recapitulate them. Although in vitro two-dimensional (2D) human-specific models have been highly used to address some of these limitations, they lack crucial features such as complexity and heterogeneity. In this review, we will discuss the advantages, limitations and future applications of in vivo and in vitro models that are used today to model NDDs. Additionally, we will describe the recent development of 3-dimensional brain (3D) organoids which offer a promising approach as human-specific in vitro models to decipher these complex disorders.

Perturbation and mechanistic studies have shown that the DNA-binding transcription factor Zeb2 controls cell fate decision, differentiation and/or maturation in multiple cell lineages in embryos and after birth. In cultured embryonic stem cells (ESCs) Zeb2 strong upregulation is necessary for their exit from primed pluripotency and entering neural and general differentiation. We engineered mouse ESCs to produce epitope-tagged Zeb2 from one of its two endogenous alleles. Using crosslinking ChIP-sequencing, we mapped for the first time 2,432 DNA-binding sites of Zeb2 in ESC-derived neuroprogenitor cells (NPCs). A new, major site maps promoter-proximal to Zeb2 itself, and its homozygous removal demonstrates that Zeb2 autoregulation is necessary to elicit proper Zeb2 effects in ESC toNPC differentiation. We then cross-referenced all Zeb2 DNA-binding sites with transcriptome data from Zeb2 perturbations in ESCs, ventral forebrain in mouse embryos, and adult neurogenesis. While the characteristics of these neurodevelopmental systems differ, we find interesting overlaps. Collectively, these new results obtained in ESC-derived NPCs significantly add to Zeb2 role as neurodevelopmental regulator as well as the causal gene in Mowat-Wilson Syndrome. Also, Zeb2 was found to map to loci mutated in other human congenital syndromes, making variant or disturbed levels of ZEB2 a candidate modifier principle in these.