Smad6 promotes neuronal differentiation in the intermediate zone of the dorsal neural tube by inhibition of the Wnt/β-catenin pathway

Abstract

Proliferation of the neural/neuronal progenitor cells (NPCs) at the ventricular zone of the dorsal spinal cord requires the stimuli of Wnt and bone morphogenic protein (BMP). However, how these two signaling pathways are regulated to initiate differentiation in the NPCs as they enter the intermediate zone is not known. Here, we show that Smad6, a negative regulator of BMP signaling, is expressed in the intermediate zone of the chick dorsal spinal cord. Knockdown experiments show that Smad6 is required for promoting NPCs to exit the cell cycle and differentiate into neurons. Although we find that Smad6 inhibits BMP signaling, as expected, we also find that Smad6 unexpectedly inhibits the Wnt/β-catenin pathway. The inhibition of the Wnt/β-catenin pathway by Smad6 is independent of its effect on the BMP pathway. Rather, Smad6 through its N-terminal domain and link region enhances the interaction of C-terminal binding protein with the β-catenin/T cell factor (TCF) complex and the TCF-binding element to inhibit β-cateninmediated transcriptional activation. Our study provides evidence that transition of NPCs from a proliferative state to a differentiating state is controlled by the dual inhibitory role of Smad6 to both BMP and Wnt signaling at the level of transcription.intermediate zone | neurogenesis P recise spatial and temporal regulation of neural development is essential for generating the complex central nervous system (CNS) found in adult organisms (1, 2). In early neural development, neural progenitor cells are seemingly homogeneous (3, 4); as neurogenesis proceeds, cells acquire different developmental potentials and become located in different tissue compartments or zones (5). At midneurogenesis, the developing cerebral cortex is composed of the ventricular zone (VZ), subventricular zone (SVZ), intermediate zone (IZ), cortical plate (CP), and marginal zone (6). Cells located in the VZ are proliferative neural progenitor cells (7). These cells progressively initiate the expression of proneural genes, such as mouse achaete-scute complex homolog 1 (Mash1) and neurogenins in response to region-specific neurogenic signals (8-10). Proneural genes drive neural progenitor cells to become intermediate neuronal progenitor cells, and these cells migrate out from the VZ and localize to the SVZ (11, 12). Subsequently, neuronal progenitor cells exit the cell cycle, differentiate into nascent neurons in the IZ, and then migrate to the CP (11). It is generally accepted that in the developing spinal cord proliferative neural/ neuronal progenitor cells (NPCs) in the VZ produce postmitotic neurons that migrate directly into the mantle zone (MZ). However, in the developing chick spinal cord, a transition region similar to the IZ has been described between the VZ and MZ marked by expression of the neurogenic differentiation 4 (NeuroM) transcription factor at midneurogenesis (13). It is not clear whether other genes also are expressed in a similar pattern and are potentially involved in the transition between NPC proliferation a...

Canonical BMP7 activity is required for the generation of discrete neuronal populations in the dorsal spinal cord

“…On the one hand, Xie and colleagues (Xie et al, 2011) found that, in the dorsal spinal cord, Smad6 promotes neuronal differentiation by repressing the Wnt canonical pathway, and thus resulting in the reduced proliferation rate of neural progenitors. On the other hand, Hazen and colleagues (Hazen et al, 2011) showed that electroporation of Smad6 or Smad7, at the time of dIN neurogenesis, results in a reduction of dI1 and dI3, without the compensatory increase of dI4 neurons.…”

C-terminal binding proteins: central players in development and disease

“…For instance, emerging neural crest cells express CtBP2, whereas dorsal root ganglia express CtBP1 (84). The importance of CtBP expression during avian CNS development is highlighted by the key involvement of these proteins in regulating the transition of neural precursor cells in the ventricular zone of the dorsal spinal cord from a proliferative to a differentiated state (85). Furthermore, as previously mentioned, CtBPs appear to have an important role in neural tube closure and development of the forebrain and hindbrain in mice (6).…”

Smad2 and Smad3 cooperate and antagonize simultaneously in vertebrate neurogenesis

“…Thus, monitoring the position of electroporated cells by evaluating EGFP expression together with that of HuC/D serves to assess neurogenesis in vivo ( Fig. 2A) (García-Campmany and Martí, 2007;Xie et al, 2011). When analyzed 48 hours post electroporation (hpe) of either control (pCIG) or Smad2, EGFP + cells were evenly distributed throughout the ventricular zone and the mantle zone (HuC/D + cells: Fig.…”