No abstract

Directed differentiation of pluripotent stem cells (PSCs) is a powerful model system for deconstructing embryonic development. Although mice are the most advanced mammalian model system for genetic studies of embryonic development, state-of-the-art protocols for directed differentiation of mouse PSCs into defined lineages require additional steps and generate target cell types with lower purity than analogous protocols for human PSCs, limiting their application as models for mechanistic studies of development. Here, we examine the potential of mouse epiblast stem cells (EpiSCs) cultured in media containing Wnt pathway inhibitors as a starting point for directed differentiation. As a proof-of-concept, we focused our efforts on two specific cell/tissue types that have proven difficult to generate efficiently and reproducibly from mouse embryonic stem cells: definitive endoderm and neural organoids. We present new protocols for rapid generation of nearly pure definitive endoderm and forebrain-patterned neural organoids that model the development of prethalamic and hippocampal neurons. These differentiation models present new possibilities for combining mouse genetic tools with in vitro differentiation to characterize molecular and cellular mechanisms of embryonic development.

CCCTC-binding factor (CTCF) and MAZ are recognized insulators required for shielding repressed posterior genes from active anterior genes within the Hox clusters during motor neuron (MN) differentiation. CTCF and MAZ interact independently with cohesin and regulate three-dimensional genome organization. Here, we followed cohesin re-location upon CTCF and MAZ depletion in mouse embryonic stem cells (mESCs) to identify novel insulators. Cohesin relocated to DNA motifs for various transcription factors, including PATZ1 and other zinc finger proteins (ZNFs). Moreover, PATZ1 and ZNFs co-localized with CTCF, MAZ, and cohesin with apparent overlapping specificity as dictated by the site to be insulated. Similar to CTCF and MAZ, PATZ1 interacted with RAD21. Patz1 KO mESCs exhibited altered global gene expression. While the absence of MAZ impacts anterior CTCF-boundaries as shown previously1, Patz1 KO led to de-repression of posterior Hox genes, resulting in cervicothoracic transformation of motor neuron (MN) fate during differentiation. These findings point to a varied, combinatorial binding of known and newly defined accessory factors as being critical for positional identity and cellular fate determination during differentiation.