Multifaceted actions of Zeb2 in postnatal neurogenesis from the ventricular-subventricular zone to the olfactory bulb.

The transcription factor Zeb2 controls fate specification and subsequent differentiation and maturation of multiple cell types in various embryonic tissues. It binds many protein partners, including activated Smad proteins and the NuRD co-repressor complex. How Zeb2 subdomains support cell differentiation in various contexts has remained elusive. Here, we studied the role of Zeb2 and its domains in neurogenesis and neural differentiation in the young postnatal ventricular-subventricular zone (V-SVZ), in which neural stem cells generate olfactory bulb-destined interneurons. Conditional Zeb2 knockouts and separate acute loss- and gain-of-function approaches indicated that Zeb2 is essential for controlling apoptosis and neuronal differentiation of V-SVZ progenitors before and after birth, and we identified Sox6 as a potential downstream target gene of Zeb2. Zeb2 genetic inactivation impaired the differentiation potential of the V-SVZ niche in a cell-autonomous fashion. We also provide evidence that its normal function in the V-SVZ also involves non-autonomous mechanisms. Additionally, we demonstrate distinct roles for Zeb2 protein-binding domains, suggesting that Zeb2 partners co-determine neuronal output from the mouse V-SVZ in both quantitative and qualitative ways in early postnatal life.

Amniotic ectoderm expansion in mouse occurs via distinct modes and requires SMAD5-mediated signalling.

Upon gastrulation, the mammalian conceptus transforms rapidly from a simple bilayer into a multilayered embryo enveloped by its extra-embryonic membranes. Impaired development of the amnion, the innermost membrane, causes major malformations. To clarify the origin of the mouse amnion, we used single-cell labelling and clonal analysis. We identified four clone types with distinct clonal growth patterns in amniotic ectoderm. Two main types have progenitors in extreme proximal-anterior epiblast. Early descendants initiate and expand amniotic ectoderm posteriorly, while descendants of cells remaining anteriorly later expand amniotic ectoderm from its anterior side. Amniogenesis is abnormal in embryos deficient in the bone morphogenetic protein (BMP) signalling effector SMAD5, with delayed closure of the proamniotic canal, and aberrant amnion and folding morphogenesis. Transcriptomics of individual Smad5 mutant amnions isolated before visible malformations and tetraploid chimera analysis revealed two amnion defect sets. We attribute them to impairment of progenitors of the two main cell populations in amniotic ectoderm and to compromised cuboidal-to-squamous transition of anterior amniotic ectoderm. In both cases, SMAD5 is crucial for expanding amniotic ectoderm rapidly into a stretchable squamous sheet to accommodate exocoelom expansion, axial growth and folding morphogenesis.

Integrative and perturbation-based analysis of the transcriptional dynamics of TGFß/BMP system components in transition from embryonic stem cells to neural progenitors.

Cooperative actions of extrinsic signals and cell-intrinsic transcription factors alter gene regulatory networks enabling cells to respond appropriately to environmental cues. Signaling by transforming growth factor type ß (TGFß) family ligands (eg, bone morphogenetic proteins [BMPs] and Activin/Nodal) exerts cell-type specific and context-dependent transcriptional changes, thereby steering cellular transitions throughout embryogenesis. Little is known about coordinated regulation and transcriptional interplay of the TGFß system. To understand intrafamily transcriptional regulation as part of this system's actions during development, we selected 95 of its components and investigated their mRNA-expression dynamics, gene-gene interactions, and single-cell expression heterogeneity in mouse embryonic stem cells transiting to neural progenitors. Interrogation at 24 hour intervals identified four types of temporal gene transcription profiles that capture all stages, that is, pluripotency, epiblast formation, and neural commitment. Then, between each stage we performed esiRNA-based perturbation of each individual component and documented the effect on steady-state mRNA levels of the remaining 94 components. This exposed an intricate system of multilevel regulation whereby the majority of gene-gene interactions display a marked cell-stage specific behavior. Furthermore, single-cell RNA-profiling at individual stages demonstrated the presence of detailed co-expression modules and subpopulations showing stable co-expression modules such as that of the core pluripotency genes at all stages. Our combinatorial experimental approach demonstrates how intrinsically complex transcriptional regulation within a given pathway is during cell fate/state transitions.

Zeb2 Regulates Myogenic Differentiation in Pluripotent Stem Cells.

Skeletal muscle differentiation is triggered by a unique family of myogenic basic helix-loop-helix transcription factors, including MyoD, MRF-4, Myf-5, and Myogenin. These transcription factors bind promoters and distant regulatory regions, including E-box elements, of genes whose expression is restricted to muscle cells. Other E-box binding zinc finger proteins target the same DNA response elements, however, their function in muscle development and regeneration is still unknown. Here, we show that the transcription factor zinc finger E-box-binding homeobox 2 (Zeb2, Sip-1, Zfhx1b) is present in skeletal muscle tissues. We investigate the role of Zeb2 in skeletal muscle differentiation using genetic tools and transgenic mouse embryonic stem cells, together with single-cell RNA-sequencing and in vivo muscle engraftment capability. We show that Zeb2 over-expression has a positive impact on skeletal muscle differentiation in pluripotent stem cells and adult myogenic progenitors. We therefore propose that Zeb2 is a novel myogenic regulator and a possible target for improving skeletal muscle regeneration. The non-neural roles of Zeb2 are poorly understood.

p120 Catenin-Mediated Stabilization of E-Cadherin Is Essential for Primitive Endoderm Specification.

E-cadherin-mediated cell-cell adhesion is critical for naive pluripotency of cultured mouse embryonic stem cells (mESCs). E-cadherin-depleted mESC fail to downregulate their pluripotency program and are unable to initiate lineage commitment. To further explore the roles of cell adhesion molecules during mESC differentiation, we focused on p120 catenin (p120ctn). Although one key function of p120ctn is to stabilize and regulate cadherin-mediated cell-cell adhesion, it has many additional functions, including regulation of transcription and Rho GTPase activity. Here, we investigated the role of mouse p120ctn in early embryogenesis, mESC pluripotency and early fate determination. In contrast to the E-cadherin-null phenotype, p120ctn-null mESCs remained pluripotent, but their in vitro differentiation was incomplete. In particular, they failed to form cystic embryoid bodies and showed defects in primitive endoderm formation. To pinpoint the underlying mechanism, we undertook a structure-function approach. Rescue of p120ctn-null mESCs with different p120ctn wild-type and mutant expression constructs revealed that the long N-terminal domain of p120ctn and its regulatory domain for RhoA were dispensable, whereas its armadillo domain and interaction with E-cadherin were crucial for primitive endoderm formation. We conclude that p120ctn is not only an adaptor and regulator of E-cadherin, but is also indispensable for proper lineage commitment.

Zeb2 Regulates Cell Fate at the Exit from Epiblast State in Mouse Embryonic Stem Cells.

In human embryonic stem cells (ESCs) the transcription factor Zeb2 regulates neuroectoderm versus mesendoderm formation, but it is unclear how Zeb2 affects the global transcriptional regulatory network in these cell-fate decisions. We generated Zeb2 knockout (KO) mouse ESCs, subjected them as embryoid bodies (EBs) to neural and general differentiation and carried out temporal RNA-sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS) analysis in neural differentiation. This shows that Zeb2 acts preferentially as a transcriptional repressor associated with developmental progression and that Zeb2 KO ESCs can exit from their naïve state. However, most cells in these EBs stall in an early epiblast-like state and are impaired in both neural and mesendodermal differentiation. Genes involved in pluripotency, epithelial-to-mesenchymal transition (EMT), and DNA-(de)methylation, including Tet1, are deregulated in the absence of Zeb2. The observed elevated Tet1 levels in the mutant cells and the knowledge of previously mapped Tet1-binding sites correlate with loss-of-methylation in neural-stimulating conditions, however, after the cells initially acquired the correct DNA-methyl marks. Interestingly, cells from such Zeb2 KO EBs maintain the ability to re-adapt to 2i + LIF conditions even after prolonged differentiation, while knockdown of Tet1 partially rescues their impaired differentiation. Hence, in addition to its role in EMT, Zeb2 is critical in ESCs for exit from the epiblast state, and links the pluripotency network and DNA-methylation with irreversible commitment to differentiation. Stem Cells 2017;35:611-625.

MTG8 interacts with LHX6 to specify cortical interneuron subtype identity.

Cortical interneurons originating in the embryonic medial ganglionic eminence (MGE) diverge into a range of different subtypes found in the adult mouse cerebral cortex. The mechanisms underlying this divergence and the timing when subtype identity is set up remain unclear. We identify the highly conserved transcriptional co-factor MTG8 as being pivotal in the development of a large subset of MGE cortical interneurons that co-expresses Somatostatin (SST) and Neuropeptide Y (NPY). MTG8 interacts with the pan-MGE transcription factor LHX6 and together the two factors are sufficient to promote expression of critical cortical interneuron subtype identity genes. The SST-NPY cortical interneuron fate is initiated early, well before interneurons migrate into the cortex, demonstrating an early onset specification program. Our findings suggest that transcriptional co-factors and modifiers of generic lineage specification programs may hold the key to the emergence of cortical interneuron heterogeneity from the embryonic telencephalic germinal zones.

Transient developmental imbalance of cortical interneuron subtypes presages long-term changes in behavior.

Cortical GABAergic interneurons are generated in large numbers in the ganglionic eminences and migrate into the cerebral cortex during embryogenesis. At early postnatal stages, during neuronal circuit maturation, autonomous and activity-dependent mechanisms operate within the cortex to adjust cell numbers by eliminating naturally occurring neuron excess. Here, we show that when cortical interneurons are generated in aberrantly high numbers-due to a defect in precursor cell proliferation during embryogenesis-extra parvalbumin interneurons persist in the postnatal mouse cortex during critical periods of cortical network maturation. Even though cell numbers are subsequently normalized, behavioral abnormalities remain in adulthood. This suggests that timely clearance of excess cortical interneurons is critical for correct functional maturation of circuits that drive adult behavior.

Transcriptional repressor ZEB2 promotes terminal differentiation of CD8+ effector and memory T cell populations during infection.

ZEB2 is a multi-zinc-finger transcription factor known to play a significant role in early neurogenesis and in epithelial-mesenchymal transition-dependent tumor metastasis. Although the function of ZEB2 in T lymphocytes is unknown, activity of the closely related family member ZEB1 has been implicated in lymphocyte development. Here, we find that ZEB2 expression is up-regulated by activated T cells, specifically in the KLRG1(hi) effector CD8(+) T cell subset. Loss of ZEB2 expression results in a significant loss of antigen-specific CD8(+) T cells after primary and secondary infection with a severe impairment in the generation of the KLRG1(hi) effector memory cell population. We show that ZEB2, which can bind DNA at tandem, consensus E-box sites, regulates gene expression of several E-protein targets and may directly repress Il7r and Il2 in CD8(+) T cells responding to infection. Furthermore, we find that T-bet binds to highly conserved T-box sites in the Zeb2 gene and that T-bet and ZEB2 regulate similar gene expression programs in effector T cells, suggesting that T-bet acts upstream and through regulation of ZEB2. Collectively, we place ZEB2 in a larger transcriptional network that is responsible for the balance between terminal differentiation and formation of memory CD8(+) T cells.

Four amino acids within a tandem QxVx repeat in a predicted extended α-helix of the Smad-binding domain of Sip1 are necessary for binding to activated Smad proteins.

The zinc finger transcription factor Smad-interacting protein-1 (Sip1; Zeb2, Zfhx1b) plays an important role during vertebrate embryogenesis in various tissues and differentiating cell types, and during tumorigenesis. Previous biochemical analysis suggests that interactions with several partner proteins, including TGFß family receptor-activated Smads, regulate the activities of Sip1 in the nucleus both as a DNA-binding transcriptional repressor and activator. Using a peptide aptamer approach we mapped in Sip1 its Smad-binding domain (SBD), initially defined as a segment of 51 amino acids, to a shorter stretch of 14 amino acids within this SBD. Modelling suggests that this short SBD stretch is part of an extended α-helix that may fit the binding to a hydrophobic corridor within the MH2 domain of activated Smads. Four amino acids (two polar Q residues and two non-polar V residues) that form the tandem repeat (QxVx)2 in this 14-residue stretch were found to be crucial for binding to both TGFß/Nodal/Activin-Smads and BMP-Smads. A full-length Sip1 with collective mutation of these Q and V residues (to A) no longer binds to Smads, while it retains its binding activity to its cognate bipartite target DNA sequence. This missense mutant Sip1(AxAx)2 provides a new molecular tool to identify SBD (in)dependent target genes in Sip1-controlled TGFß and/or BMP (de)regulated cellular, developmental and pathological processes.

Few Smad proteins and many Smad-interacting proteins yield multiple functions and action modes in TGFß/BMP signaling in vivo.

Signaling by the many ligands of the TGFß family strongly converges towards only five receptor-activated, intracellular Smad proteins, which fall into two classes i.e. Smad2/3 and Smad1/5/8, respectively. These Smads bind to a surprisingly high number of Smad-interacting proteins (SIPs), many of which are transcription factors (TFs) that co-operate in Smad-controlled target gene transcription in a cell type and context specific manner. A combination of functional analyses in vivo as well as in cell cultures and biochemical studies has revealed the enormous versatility of the Smad proteins. Smads and their SIPs regulate diverse molecular and cellular processes and are also directly relevant to development and disease. In this survey, we selected appropriate examples on the BMP-Smads, with emphasis on Smad1 and Smad5, and on a number of SIPs, i.e. the CPSF subunit Smicl, Ttrap (Tdp2) and Sip1 (Zeb2, Zfhx1b) from our own research carried out in three different vertebrate models.

Sip1 regulates sequential fate decisions by feedback signaling from postmitotic neurons to progenitors.

The fate of cortical progenitors, which progressively generate neurons and glial cells during development, is determined by temporally and spatially regulated signaling mechanisms. We found that the transcription factor Sip1 (Zfhx1b), which is produced at high levels in postmitotic neocortical neurons, regulates progenitor fate non-cell autonomously. Conditional deletion of Sip1 in young neurons induced premature production of upper-layer neurons at the expense of deep layers, precocious and increased generation of glial precursors, and enhanced postnatal astrocytogenesis. The premature upper-layer generation coincided with overexpression of the neurotrophin-3 (Ntf3) gene and upregulation of fibroblast growth factor 9 (Fgf9) gene expression preceded precocious gliogenesis. Exogenous application of Fgf9 to mouse cortical slices induced excessive generation of glial precursors in the germinal zone. Our data suggest that Sip1 restrains the production of signaling factors in postmitotic neurons that feed back to progenitors to regulate the timing of cell fate switch and the number of neurons and glial cells throughout corticogenesis.