Introduction:

Specific inactivation of TGF␤ signaling in neural crest stem cells (NCSCs) results in cardiovascular defects and thymic, parathyroid, and craniofacial anomalies. All these malformations characterize DiGeorge syndrome, the most common microdeletion syndrome in humans. Consistent with a role of TGF␤ in promoting non-neural lineages in NCSCs, mutant neural crest cells migrate into the pharyngeal apparatus but are unable to acquire non-neural cell fates. Moreover, in neural crest cells, TGF␤ signaling is both sufficient and required for phosphorylation of CrkL, a signal adaptor protein implicated in the development of DiGeorge syndrome. Thus, TGF␤ signal modulation in neural crest differentiation might play a crucial role in the etiology of DiGeorge syndrome.Supplemental material is available at http://www.genesdev.org.Received July 16, 2004; revised version accepted December 23, 2004. During development, neural crest cells emerge from the dorsal part of the neural tube and emigrate to various locations within the embryo to generate most of the peripheral nervous system and a variety of other structures (Le Douarin and Dupin 2003). In particular, neural crest cells localized in the pharyngeal apparatus contribute to non-neural tissues, such as craniofacial bone and cartilage, thymus, parathyroid glands, and cardiac outflow tract and septum (Kirby and Waldo 1995;Jiang et al. 2000;Graham 2003). The function of neural crest cells in the generation of these tissues, however, has been debated (Graham 2003).Formation of the pharyngeal apparatus involves complex interactions of neural crest, ectoderm, endoderm, and mesoderm whose development must be coordinated (Graham 2003). Significantly, alterations to the development of this region are often associated with congenital human birth defects such as DiGeorge or Velocardiofacial syndrome. DiGeorge syndrome is the most common microdeletion syndrome in humans, characterized by cardiovascular defects plus thymic, parathyroid, and craniofacial anomalies (Lindsay 2001;Vitelli and Baldini 2003). Approximately 80% of the patients carry a variably sized deletion on chromosome 22 (del22q11). Ablation of genes affected by the microdeletion indicated two pathophysiological mechanisms causing DiGeorge syndrome: Mutations of the transcription factor Tbx1 lead to disturbed pharyngeal arch patterning. Consequently, neural crest cells are unable to populate the pharyngeal apparatus ( Previously, cell culture experiments allowed the identification of several growth factors able to instruct migratory and post-migratory neural crest cells to adopt specific lineages (Le Douarin and Dupin 2003;Lee et al. 2004). One of these factors is transforming growth factor (TGF) ␤, which elicits multiple responses in cultured neural crest stem cells (NCSCs). Depending on the cellular context, it can promote the generation of non-neural smooth-muscle-like cells or autonomic neurons, or induce apoptosis (Shah et al. 1996;Hagedorn et al. 1999Hagedorn et al. , 2000. Here we investigated the role of TGF␤ si...

Glioblastoma multiforme (GBM) is a highly aggressive form of brain cancer associated with a very poor prognosis. Recently, the initiation and growth of GBM has been linked to brain tumor-initiating cells (BTICs), which are poorly differentiated and share features with neural stem cells (NSCs). Here we describe a kinome-wide RNA interference screen to identify factors that control the tumorigenicity of BTICs. We identified several genes whose silencing induces differentiation of BTICs derived from multiple GBM patients. In particular, knockdown of the adaptor protein TRRAP significantly increased differentiation of cultured BTICs, sensitized the cells to apoptotic stimuli, and negatively affected cell cycle progression. TRRAP knockdown also significantly suppressed tumor formation upon intracranial BTIC implantation into mice. Together, these findings support a critical role for TRRAP in maintaining a tumorigenic, stem cell-like state.

Canonical Wnt signaling instructively promotes sensory neurogenesis in early neural crest stem cells (eNCSCs) (Lee, H.Y., M. Kléber, L. Hari, V. Brault, U. Suter, M.M. Taketo, R. Kemler, and L. Sommer. 2004. Science. 303:1020–1023). However, during normal development Wnt signaling induces a sensory fate only in a subpopulation of eNCSCs while other cells maintain their stem cell features, despite the presence of Wnt activity. Hence, factors counteracting Wnt signaling must exist. Here, we show that bone morphogenic protein (BMP) signaling antagonizes the sensory fate-inducing activity of Wnt/β-catenin. Intriguingly, Wnt and BMP act synergistically to suppress differentiation and to maintain NCSC marker expression and multipotency. Similar to NCSCs in vivo, NCSCs maintained in culture alter their responsiveness to instructive growth factors with time. Thus, stem cell development is regulated by combinatorial growth factor activities that interact with changing cell-intrinsic cues.

Regulating the choice between neural stem cell maintenance versus differentiation determines growth and size of the developing brain. Here we identify TGF-beta signaling as a crucial factor controlling these processes. At early developmental stages, TGF-beta signal activity is localized close to the ventricular surface of the neuroepithelium. In the midbrain, but not in the forebrain, Tgfbr2 ablation results in ectopic expression of Wnt1/beta-catenin and FGF8, activation of Wnt target genes, and increased proliferation and horizontal expansion of neuroepithelial cells due to shortened cell-cycle length and decreased cell-cycle exit. Consistent with this phenotype, self-renewal of mutant neuroepithelial stem cells is enhanced in the presence of FGF and requires Wnt signaling. Moreover, TGF-beta signal activation counteracts Wnt-induced proliferation of midbrain neuroepithelial cells. Thus, TGF-beta signaling controls the size of a specific brain area, the dorsal midbrain, by antagonizing canonical Wnt signaling and negatively regulating self-renewal of neuroepithelial stem cells.

Embryonic stem cells (ESCs) are an attractive source of cells for disease modeling in vitro and may eventually provide access to cells/tissues for the treatment of many degenerative diseases. However, applications of ESC-derived cell types are largely hindered by the lack of highly efficient methods for lineage-specific differentiation. Using a high-content screen, we have identified a small molecule, named stauprimide, that increases the efficiency of the directed differentiation of mouse and human ESCs in synergy with defined extracellular signaling cues. Affinity-based methods revealed that stauprimide interacts with NME2 and inhibits its nuclear localization. This, in turn, leads to downregulation of c-Myc, a key regulator of the pluripotent state. Thus, our findings identify a chemical tool that primes ESCs for efficient differentiation through a mechanism that affects c-Myc expression, and this study points to an important role for NME2 in ESC self-renewal.

Adult neurogenesis occurs in mammals and provides a mechanism for continuous neural plasticity in the brain. However, little is known about the molecular mechanisms regulating hippocampal neural progenitor cells (NPCs) and whether their fate can be pharmacologically modulated to improve neural plasticity and regeneration. Here, we report the characterization of a small molecule (KHS101) that selectively induces a neuronal differentiation phenotype. Mechanism of action studies revealed a link of KHS101 to cell cycle exit and specific binding to the TACC3 protein, whose knockdown in NPCs recapitulates the KHS101-induced phenotype. Upon systemic administration, KHS101 distributed to the brain and resulted in a significant increase in neuronal differentiation in vivo. Our findings indicate that KHS101 accelerates neuronal differentiation by interaction with TACC3 and may provide a basis for pharmacological intervention directed at endogenous NPCs.

Multiple signaling pathways regulate proliferation and differentiation of neural progenitor cells during early development of the central nervous system (CNS). In the spinal cord, dorsal signaling by bone morphogenic protein (BMP) acts primarily as a patterning signal, while canonical Wnt signaling promotes cell cycle progression in stem and progenitor cells. However, overexpression of Wnt factors or, as shown here, stabilization of the Wnt signaling component beta-catenin has a more prominent effect in the ventral than in the dorsal spinal cord, revealing local differences in signal interpretation. Intriguingly, Wnt signaling is associated with BMP signal activation in the dorsal spinal cord. This points to a spatially restricted interaction between these pathways. Indeed, BMP counteracts proliferation promoted by Wnt in spinal cord neuroepithelial cells. Conversely, Wnt antagonizes BMP-dependent neuronal differentiation. Thus, a mutually inhibitory crosstalk between Wnt and BMP signaling controls the balance between proliferation and differentiation. A model emerges in which dorsal Wnt/BMP signal integration links growth and patterning, thereby maintaining undifferentiated and slow-cycling neural progenitors that form the dorsal confines of the developing spinal cord.