Bennett G. Novitch

Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD

Skeletal muscle differentiation entails the coordination of muscle-specific gene expression and terminal withdrawal from the cell cycle. This cell cycle arrest in the G0 phase requires the retinoblastoma tumor suppressor protein (Rb). The function of Rb is negatively regulated by cyclin-dependent kinases (Cdks), which are controlled by Cdk inhibitors. Expression of MyoD, a skeletal muscle-specific transcriptional regulator, activated the expression of the Cdk inhibitor p21 during differentiation of murine myocytes and in nonmyogenic cells. MyoD-mediated induction of p21 did not require the tumor suppressor protein p53 and correlated with cell cycle withdrawal. Thus, MyoD may induce terminal cell cycle arrest during skeletal muscle differentiation by increasing the expression of p21.

Vertebrate neurogenesis is counteracted by Sox1-3 activity.

The generation of neurons from stem cells involves the activity of proneural basic helix-loop-helix (bHLH) proteins, but the mechanism by which these proteins irreversibly commit stem cells to neuronal differentiation is not known. Here we report that expression of the transcription factors Sox1, Sox2 and Sox3 (Sox1–3) is a critical determinant of neurogenesis. Using chick in ovo electroporation, we found that Sox1–3 transcription factors keep neural cells undifferentiated by counteracting the activity of proneural proteins. Conversely, the capacity of proneural bHLH proteins to direct neuronal differentiation critically depends on their ability to suppress Sox1–3 expression in CNS progenitors. These data suggest that the generation of neurons from stem cells depends on the inhibition of Sox1–3 expression by proneural proteins.

Coordinate Regulation of Motor Neuron Subtype Identity and Pan-Neuronal Properties by the bHLH Repressor Olig2

Within the developing vertebrate nervous system, the mechanisms that coordinate neuronal subtype identity with generic features of neuronal differentiation are poorly defined. We show here that a bHLH protein, Olig2, is expressed selectively by motor neuron progenitors and has a key role in specifying the subtype identity and pan-neuronal properties of developing motor neurons. The role of Olig2 in the specification of motor neuron subtype identity depends on regulatory interactions with progenitor homeodomain proteins, whereas its role in promoting pan-neuronal properties is associated with expression of another bHLH protein, Ngn2. Both aspects of Olig2 function appear to depend on its activity as a transcriptional repressor. Together, these studies show that Olig2 has a critical role in integrating diverse features of motor neuron differentiation in the developing spinal cord.

Interpretation of the sonic hedgehog morphogen gradient by a temporal adaptation mechanism

Morphogens act in developing tissues to control the spatial arrangement of cellular differentiation. The activity of a morphogen has generally been viewed as a concentration-dependent response to a diffusible signal, but the duration of morphogen signalling can also affect cellular responses. One such example is the morphogen sonic hedgehog (SHH). In the vertebrate central nervous system and limbs, the pattern of cellular differentiation is controlled by both the amount and the time of SHH exposure. How these two parameters are interpreted at a cellular level has been unclear. Here we provide evidence that changing the concentration or duration of SHH has an equivalent effect on intracellular signalling. Chick neural cells convert different concentrations of SHH into time-limited periods of signal transduction, such that signal duration is proportional to SHH concentration. This depends on the gradual desensitization of cells to ongoing SHH exposure, mediated by the SHH-dependent upregulation of patched 1 (PTC1), a ligand-binding inhibitor of SHH signalling. Thus, in addition to its role in shaping the SHH gradient, PTC1 participates cell autonomously in gradient sensing. Together, the data reveal a novel strategy for morphogen interpretation, in which the temporal adaptation of cells to a morphogen integrates the concentration and duration of a signal to control differential gene expression.

Directed differentiation of human induced pluripotent stem cells generates active motor neurons

The potential for directed differentiation of human‐induced pluripotent stem (iPS) cells to functional postmitotic neuronal phenotypes is unknown. Following methods shown to be effective at generating motor neurons from human embryonic stem cells (hESCs), we found that once specified to a neural lineage, human iPS cells could be differentiated to form motor neurons with a similar efficiency as hESCs. Human iPS‐derived cells appeared to follow a normal developmental progression associated with motor neuron formation and possessed prototypical electrophysiological properties. This is the first demonstration that human iPS‐derived cells are able to generate electrically active motor neurons. These findings demonstrate the feasibility of using iPS‐derived motor neuron progenitors and motor neurons in regenerative medicine applications and in vitro modeling of motor neuron diseases. STEM CELLS 2009;27:806–811

Regulatory mechanisms that coordinate skeletal muscle differentiation and cell cycle withdrawal

Skeletal muscle differentiation entails the coupling of muscle-specific gene expression to terminal withdrawal from the cell cycle. Several models have recently been proposed which attempt to explain how regulated expression and function of myogenic transcription factors ensures that proliferation and differentiation of skeletal muscle cells are mutually exclusive processes.

A Requirement for Retinoic Acid-Mediated Transcriptional Activation in Ventral Neural Patterning and Motor Neuron Specification

The specification of neuronal fates in the ventral spinal cord depends on the regulation of homeodomain (HD) and basic-helix-loop-helix (bHLH) proteins by Sonic hedgehog (Shh). Most of these transcription factors function as repressors, leaving unresolved the link between inductive signaling pathways and transcriptional activators involved in ventral neuronal specification. We show here that retinoid signaling and the activator functions of retinoid receptors are required to pattern the expression of HD and bHLH proteins and to specify motor neuron identity. We also show that fibroblast growth factors (FGFs) repress progenitor HD protein expression, implying that evasion of FGF signaling and exposure to retinoid and Shh signals are obligate steps in the emergence of ventral neural pattern. Moreover, joint exposure of neural progenitors to retinoids and FGFs suffices to induce motor neuron differentiation in a Shh-independent manner.

Coordinated Actions of the Forkhead Protein Foxp1 and Hox Proteins in the Columnar Organization of Spinal Motor Neurons

The formation of locomotor circuits depends on the spatially organized generation of motor columns that innervate distinct muscle and autonomic nervous system targets along the body axis. Within each spinal segment, multiple motor neuron classes arise from a common progenitor population; however, the mechanisms underlying their diversification remain poorly understood. Here, we show that the Forkhead domain transcription factor Foxp1 plays a critical role in defining the columnar identity of motor neurons at each axial position. Using genetic manipulations, we demonstrate that Foxp1 establishes the pattern of LIM-HD protein expression and accordingly organizes motor axon projections, their connectivity with peripheral targets, and the establishment of motor pools. These functions of Foxp1 act in accordance with the rostrocaudal pattern provided by Hox proteins along the length of the spinal cord, suggesting a model by which motor neuron diversity is achieved through the coordinated actions of Foxp1 and Hox proteins.

pRb is required for MEF2-dependent gene expression as well as cell-cycle arrest during skeletal muscle differentiation.

BACKGROUND The onset of differentiation-specific gene expression in skeletal muscle is coupled to permanent withdrawal from the cell cycle. The retinoblastoma tumor-suppressor protein (pRb) is a critical regulator of this process, required for both cell-cycle arrest in G0 phase and high-level expression of late muscle-differentiation markers. Although the cell-cycle defects that are seen in pRb-deficient myocytes can be explained by the well-described function of pRb as a negative regulator of the transition from G1 to S phase, it remains unclear how pRb positively affects late muscle-gene expression. RESULTS Here, we show that the myogenic defect in Rb-/- cells corresponds to a deficiency in the activity of the transcription factor MEF2. Without pRb, MyoD induces the accumulation of nuclear-localized MEF2 that is competent to bind DNA yet transcriptionally inert. When pRb is present, MyoD stimulates the function of the MEF2C transcriptional activation domain and the activity of endogenous MEF2-type factors. Co-transfection of MyoD together with an activated form of MEF2C containing the Herpesvirus VP16 transcriptional activation domain partially bypasses the requirement for pRb and induces late muscle-gene expression in replicating cells. This ectopic myogenesis is nevertheless significantly augmented by co-expression of an E2F1-pRb chimeric protein that blocks the cell cycle. CONCLUSION These findings indicate that pRb promotes the expression of late-stage muscle-differentiation markers by both inhibiting cell-cycle progression and cooperating with MyoD to promote the transcriptional activation activity of MEF2.

Olig2 Directs Astrocyte and Oligodendrocyte Formation in Postnatal Subventricular Zone Cells

The subventricular zone (SVZ) in the neonatal mammalian forebrain simultaneously generates olfactory interneurons, astrocytes, and oligodendrocytes. The molecular cues that enable SVZ progenitors to generate three distinct cell lineages without a temporal switching mechanism are not known. Here, we demonstrate that the basic helix-loop-helix transcription factor Olig2 plays a central role in this process. Olig2 is specifically expressed in gliogenic progenitors in the postnatal SVZ and by all glial lineages derived from this structure. By expressing normal and dominant-interfering forms of Olig2 in vivo, we show that Olig2 repressor function is both sufficient and necessary to prevent neuronal differentiation and to direct SVZ progenitors toward astrocytic and oligodendrocytic fates. Although Olig2 activity has been associated previously with motor neuron and oligodendrocyte development, our findings establish a previously unappreciated role for Olig2 in the development of astrocytes. Furthermore, these results indicate that Olig2 serves a critical role in pan-glial versus neuronal fate decisions in SVZ progenitors, making it the first intrinsic fate determinant shown to operate in the early postnatal SVZ.