Susanne J. Kühl

On the role of Wnt/β-catenin signaling in stem cells.

BACKGROUND Stem cells are mainly characterized by two properties: self-renewal and the potency to differentiate into diverse cell types. These processes are regulated by different growth factors including members of the Wnt protein family. Wnt proteins are secreted glycoproteins that can activate different intracellular signaling pathways. SCOPE OF REVIEW Here we summarize our current knowledge on the role of Wnt/β-catenin signaling with respect to these two main features of stem cells. MAJOR CONCLUSIONS A particular focus is given on the function of Wnt signaling in embryonic stem cells. Wnt signaling can also improve reprogramming of somatic cells towards iPS cells highlighting the importance of this pathway for self-renewal and pluripotency. As an example for the role of Wnt signaling in adult stem cell behavior, we furthermore focus on intestinal stem cells located in the crypts of the small intestine. GENERAL SIGNIFICANCE A broad knowledge about stem cell properties and the influence of intrinsic and extrinsic factors on these processes is a requirement for the use of these cells in regenerative medicine in the future or to understand cancer development in the adult. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

TBX3 Directs Cell-Fate Decision toward Mesendoderm

Summary Cell-fate decisions and pluripotency are dependent on networks of key transcriptional regulators. Recent reports demonstrated additional functions of pluripotency-associated factors during early lineage commitment. The T-box transcription factor TBX3 has been implicated in regulating embryonic stem cell self-renewal and cardiogenesis. Here, we show that TBX3 is dynamically expressed during specification of the mesendoderm lineages in differentiating embryonic stem cells (ESCs) in vitro and in developing mouse and Xenopus embryos in vivo. Forced TBX3 expression in ESCs promotes mesendoderm specification by directly activating key lineage specification factors and indirectly by enhancing paracrine Nodal/Smad2 signaling. TBX3 loss-of-function analyses in the Xenopus underline its requirement for mesendoderm lineage commitment. Moreover, we uncovered a functional redundancy between TBX3 and Tbx2 during Xenopus gastrulation. Taken together, we define further facets of TBX3 actions and map TBX3 as an upstream regulator of the mesendoderm transcriptional program during gastrulation.

Lypd6 Enhances Wnt/β-Catenin Signaling by Promoting Lrp6 Phosphorylation in Raft Plasma Membrane Domains

Wnt/β-catenin signaling plays critical roles during embryogenesis, tissue homeostasis, and regeneration. How Wnt-receptor complex activity is regulated is not yet fully understood. Here, we identify the Ly6 family protein LY6/PLAUR domain-containing 6 (Lypd6) as a positive feedback regulator of Wnt/β-catenin signaling. lypd6 enhances Wnt signaling in zebrafish and Xenopus embryos and in mammalian cells, and it is required for wnt8-mediated patterning of the mesoderm and neuroectoderm during zebrafish gastrulation. Lypd6 is GPI anchored to the plasma membrane and physically interacts with the Wnt receptor Frizzled8 and the coreceptor Lrp6. Biophysical and biochemical evidence indicates that Lypd6 preferentially localizes to raft membrane domains, where Lrp6 is phosphorylated upon Wnt stimulation. lypd6 knockdown or mislocalization of the Lypd6 protein to nonraft membrane domains shifts Lrp6 phosphorylation to these domains and inhibits Wnt signaling. Thus, Lypd6 appears to control Lrp6 activation specifically in membrane rafts, which is essential for downstream signaling.

Direct Nkx2‐5 Transcriptional Repression of Isl1 Controls Cardiomyocyte Subtype Identity

During cardiogenesis, most myocytes arise from cardiac progenitors expressing the transcription factors Isl1 and Nkx2‐5. Here, we show that a direct repression of Isl1 by Nkx2‐5 is necessary for proper development of the ventricular myocardial lineage. Overexpression of Nkx2‐5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in Isl1+ precursors. Embryos deficient for Nkx2‐5 in the Isl1+ lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube. We demonstrated that Nkx2‐5 directly binds to an Isl1 enhancer and represses Isl1 transcriptional activity. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, it leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased cardiomyocyte number. Functional and molecular characterization of Isl1‐overexpressing cardiomyocytes revealed higher beating frequencies in both ESC‐derived contracting areas and Xenopus Isl1‐gain‐of‐function hearts, which associated with upregulation of nodal‐specific genes and downregulation of transcripts of working myocardium. Immunocytochemistry of cardiomyocyte lineage‐specific markers demonstrated a reduction of ventricular cells and an increase of cells expressing the pacemaker channel Hcn4. Finally, optical action potential imaging of single cardiomyocytes combined with pharmacological approaches proved that Isl1 overexpression in ESCs resulted in normally electrophysiologically functional cells, highly enriched in the nodal subtype at the expense of the ventricular lineage. Our findings provide an Isl1/Nkx2‐5‐mediated mechanism that coordinately regulates the specification of cardiac progenitors toward the different myocardial lineages and ensures proper acquisition of myocyte subtype identity. Stem Cells 2015;33:1113–1129

Islet1-expressing cardiac progenitor cells: a comparison across species.

Adult mammalian cardiac stem cells express the LIM-homeodomain transcription factor Islet1 (Isl1). They are considered remnants of Isl1-positive embryonic cardiac progenitor cells. During amniote heart development, Isl1-positive progenitor cells give rise mainly to the outflow tract, the right ventricle, and parts of the atria. This led to the hypothesis that the development of the right ventricle of the amniote heart depends on the recruitment of additional cells to the primary heart tube. The region from which these additional, Isl1-positive cells originate is called second heart field, as opposed to the first heart field whose cells form the primary heart tube. Here, we review the available data about Isl1 in different species, demonstrating that Isl1 is an important component of the core transcription factor network driving early cardiogenesis in animals of the two clades, deuterostomes, and protostomes. The data support the view of a single cardiac progenitor cell population that includes Isl1-expressing cells and which differentiates into the various cardiac lineages during embryonic development in vertebrates but not in other phyla of the animal kingdom.

WT1 and Sox11 regulate synergistically the promoter of the Wnt4 gene that encodes a critical signal for nephrogenesis

Wnt4, a member of the Wnt superfamily of signaling molecules, is critical for mammalian kidney development, since nephrogenesis fails in its absence, while Wnt4 signaling induces mesenchyme-to-epithelium transition and associated tubulogenesis in the uninduced mesenchymal cells in the classic transfilter model. The factors that promote Wnt4 gene expression during kidney development are largely unknown, however. We addressed the upstream regulators of the Wnt4 gene and describe here the transcription factors WT1 and Sox11 as candidate molecules in the control of gene expression. We found that WT1/Sox11 regulate Wnt4 promoter expression in a synergistic fashion in an embryonic kidney mesenchyme-derived cell line model. The transcription complex containing WT1/Sox11 was immunoprecipitated from embryonic kidney cells with Sox11 antibodies, suggesting their presence in the same complex. Dominant negative forms of WT1, namely P129L and F154S mutants, inhibited Wnt4 expression, but this inhibition was not influenced by the presence of wild-type Sox11. The mutant WT1 forms were similarly incapable of interacting with Sox11, as judged by reporter studies. The spatio-temporal expression pattern of wt1 and sox11 overlaps with that of Wnt4 in the early Xenopus pronephros. Morpholino-mediated knockdown of either wt1 or sox11 inhibited Wnt4 expression in the prospective pronephros of the Xenopus embryos. We propose that Sox11 represents a synergistic factor for WT1 in regulating the Wnt4 gene expression that is critical for nephrogenesis during kidney ontogeny.

Tbx5 Overexpression Favors a First Heart Field Lineage in Murine Embryonic Stem Cells and in Xenopus laevis Embryos

The T‐box transcription factor Tbx5 is involved in several developmental processes including cardiogenesis. Early steps of cardiac development are characterised by the formation of two cardiogenic lineages, the first (FHF) and the second heart field (SHF) lineage, which arise from a common cardiac progenitor cell population. To further investigate the function of Tbx5 during cardiogenesis, we generated a murine embryonic stem cell line constitutively overexpressing Tbx5. Differentiation of these cells is characterised by an earlier and increased appearance of contracting cardiomyocytes that beat with a higher frequency than control cells. In semi‐quantitative and quantitative RT‐PCR analyses, we observed an up‐regulation of cardiac marker genes such as Troponin T, endogenous Tbx5, and Nkx2.5 and a down‐regulation of others like BMP4 and Hand2. Similar data were gained in Xenopus laevis arguing for a conserved function of Tbx5. Furthermore, markers of the conduction system and atrial cardiomyocytes were increased. Developmental Dynamics 240:2634–2645, 2011.

A phospho-dependent mechanism involving NCoR and KMT2D controls a permissive chromatin state at Notch target genes

Abstract The transcriptional shift from repression to activation of target genes is crucial for the fidelity of Notch responses through incompletely understood mechanisms that likely involve chromatin-based control. To activate silenced genes, repressive chromatin marks are removed and active marks must be acquired. Histone H3 lysine-4 (H3K4) demethylases are key chromatin modifiers that establish the repressive chromatin state at Notch target genes. However, the counteracting histone methyltransferase required for the active chromatin state remained elusive. Here, we show that the RBP-J interacting factor SHARP is not only able to interact with the NCoR corepressor complex, but also with the H3K4 methyltransferase KMT2D coactivator complex. KMT2D and NCoR compete for the C-terminal SPOC-domain of SHARP. We reveal that the SPOC-domain exclusively binds to phosphorylated NCoR. The balance between NCoR and KMT2D binding is shifted upon mutating the phosphorylation sites of NCoR or upon inhibition of the NCoR kinase CK2β. Furthermore, we show that the homologs of SHARP and KMT2D in Drosophila also physically interact and control Notch-mediated functions in vivo. Together, our findings reveal how signaling can fine-tune a committed chromatin state by phosphorylation of a pivotal chromatin-modifier.

Deletions and de novo mutations of SOX11 are associated with a neurodevelopmental disorder with features of Coffin–Siris syndrome

Background SOX11 is a transcription factor proposed to play a role in brain development. The relevance of SOX11 to human developmental disorders was suggested by a recent report of SOX11 mutations in two patients with Coffin–Siris syndrome. Here we further investigate the role of SOX11 variants in neurodevelopmental disorders. Methods We used array based comparative genomic hybridisation and trio exome sequencing to identify children with intellectual disability who have deletions or de novo point mutations disrupting SOX11. The pathogenicity of the SOX11 mutations was assessed using an in vitro gene expression reporter system. Loss-of-function experiments were performed in xenopus by knockdown of Sox11 expression. Results We identified seven individuals with chromosome 2p25 deletions involving SOX11. Trio exome sequencing identified three de novo SOX11 variants, two missense (p.K50N; p.P120H) and one nonsense (p.C29*). The biological consequences of the missense mutations were assessed using an in vitro gene expression system. These individuals had microcephaly, developmental delay and shared dysmorphic features compatible with mild Coffin–Siris syndrome. To further investigate the function of SOX11, we knocked down the orthologous gene in xenopus. Morphants had significant reduction in head size compared with controls. This suggests that SOX11 loss of function can be associated with microcephaly. Conclusions We thus propose that SOX11 deletion or mutation can present with a Coffin–Siris phenotype.

sox4 and sox11 function during Xenopus laevis eye development.

SoxC genes are involved in many developmental processes such as cardiac, lymphoid, and bone development. The SoxC gene family is represented by Sox4, Sox11, and Sox12. Loss of either Sox4 or Sox11 function is lethal during mouse embryogenesis. Here, we demonstrate that sox4 and sox11 are strongly expressed in the developing eye, heart as well as brain in Xenopus laevis. Morpholino oligonucleotide mediated knock-down approaches in anterior neural tissue revealed that interference with either Sox4 or Sox11 function affects eye development. A detailed analysis demonstrated strong effects on eye size and retinal lamination. Neural induction was unaffected upon Sox4 or Sox11 MO injection and early eye field differentiation and cell proliferation were only mildly affected. Depletion of both genes, however, led independently to a significant increase in cell apoptosis in the eye. In summary, Sox4 and Sox11 are required for Xenopus visual system development.