Nodal signaling regulates the bone morphogenic protein pluripotency pathway in mouse embryonic stem cells.

Members of the transforming growth factor-beta superfamily play essential roles in both the pluripotency and differentiation of embryonic stem (ES) cells. Although bone morphogenic proteins (BMPs) maintain pluripotency of undifferentiated mouse ES cells, the role of autocrine Nodal signaling is less clear. Pharmacological, molecular, and genetic methods were used to further understand the roles and potential interactions of these pathways. Treatment of undifferentiated ES cells with SB431542, a pharmacological inhibitor of Smad2 signaling, resulted in a rapid reduction of phosphorylated Smad2 and altered the expression of several putative downstream targets. Unexpectedly, inhibition of the Nodal signaling pathway resulted in enhanced BMP signaling, as assessed by Smad1/5 phosphorylation. SB431542-treated cells also demonstrated significant induction of the Id genes, which are known direct targets of BMP signaling and important factors in ES cell pluripotency. Inhibition of BMP signaling decreased the SB431542-mediated phosphorylation of Smad1/5 and induction of Id genes, suggesting that BMP signaling is necessary for some Smad2-mediated activity. Because Smad7, a known inhibitory factor to both Nodal and BMP signaling, was down-regulated following inhibition of Nodal-Smad2 signaling, the contribution of Smad7 to the cross-talk between the transforming growth factor-beta pathways in ES cells was examined. Biochemical manipulation of Smad7 expression, through shRNA knockdown or inducible gene expression, significantly reduced the SB431542-mediated phosphorylation of Smad1/5 and induction of the Id genes. We conclude that autocrine Nodal signaling in undifferentiated mouse ES cells modulates the vital pluripotency pathway of BMP signaling.

Gli1 induces G2/M arrest and apoptosis in hippocampal but not tumor-derived neural stem cells.

Sonic hedgehog (Shh) is necessary for sustaining the proliferation of neural stem cells (NSCs), yet little is known about its mechanisms. Whereas Gli1, Gli2, and Gli3, the primary mediators of Shh signaling, were all expressed in hippocampal neural progenitors, Shh treatment of NSCs induced only Gli1 expression. Acute depletion of Gli1 in postnatal NSCs by short-hairpin RNA decreased proliferation, whereas germline deletion of Gli1 did not affect NSC proliferation, suggesting a difference in mechanisms of Gli1 compensation that may be developmentally dependent. To determine whether Gli1 was sufficient to enhance NSC proliferation, we overexpressed this mitogen and were surprised to find that Gli1 resulted in decreased proliferation, accumulation of NSCs in the G2/M phase of cell cycle, and apoptosis. In contrast, Gli1-expressing lineage-restricted neural precursors demonstrated a 4.5-fold proliferation enhancement. Expression analyses of Gli1-expressing NSCs identified significant induction of Gadd45a and decreased cyclin A2 and Stag1 mRNA, genes involved in the G2-M transition and apoptosis. Furthermore, Gadd45a overexpression was sufficient to partially recapitulate the Gli1-induced G2/M accumulation and cell death of NSCs. In contrast to normal stem cells, tumor-derived stem cells had markedly higher basal Gli1 expression and did not undergo apoptosis with further elevation of Gli1. Our data suggest that Gli1-induced apoptosis may serve as a protective mechanism against premature mitosis and may give insight into mechanisms by which nonmalignant stem cells restrain hyperproliferation in the context of potentially transforming mitogenic signals. Tumor-derived stem cells apparently lack these mechanisms, which may contribute to their unrestrained proliferation and malignant potential.

Effects of gustatory nerve transection and regeneration on quinine-stimulated Fos-like immunoreactivity in the parabrachial nucleus of the rat.

The distribution of quinine-stimulated Fos-like immunoreactivity (FLI) in several subdivisions of the parabrachial nucleus (PBN) known to be responsive to gustatory stimulation was examined in rats in which the chorda tympani nerve (CT) and/or glossopharyngeal nerve (GL) was transected (Experiment 1) and in rats in which the GL was transected with regeneration promoted or prevented (Experiment 2). We confirmed previous findings in the literature by demonstrating that rats intraorally infused with 3 mM quinine showed a robust population of FLI in the waist area and the external lateral (EL) and external medial (EM) subdivisions of the PBN (Yamamoto et al. [1994] Physiol Behav 56:1197-1202; Travers et al., [ 1999] Am J Physiol 277:R384-R394). In the waist area, only GL transection significantly decreased the number of FLI-neurons elicited by intraoral infusion of quinine compared with water-stimulated controls. In the external subdivisions neither neurotomy affected the number of FLI-neurons. The effect of GL transection in the waist area was enduring for rats in which the GL did not regenerate (up to 94 days), but regeneration of the GL after 52 days restored quinine-stimulated FLI to control values. In these same GL-transected animals, there were parallel decreases in the number of gapes elicited by intraoral quinine stimulation that recovered, but only subsequent to regeneration of the GL. These data provide support for the role of the waist area in the brainstem processing that underlies oromotor rejection behaviors and also help substantiate the hypothesis that the CT and GL are relatively specialized with regard to function. Moreover, when the quinine-induced pattern of neural activity in the second central gustatory relay, as assessed by FLI, is substantially altered by the loss of peripheral gustatory input from the GL, it can be restored upon regeneration of the nerve.

Stimulation of specific regions of the parabrachial nucleus elicits ingestive oromotor behaviors in conscious rats.

The "waist" area and external subnuclei of the parabrachial nucleus (PBN) have been implicated in the processing of gustatory information, yet their behavioral roles are not clearly defined. In the current study, areas within and surrounding the PBN were stimulated while oromotor behaviors were monitored in conscious rats. Electrical and chemical (100 mM glutamate) stimulation of the waist area increased ingestive oromotor behaviors over baseline (p<.01). Stimulation of external PBN subnuclei and areas medial and ventral to the PBN failed to cause a behavioral change. These data support the hypothesis that the waist area of the PBN constitutes part of the neural substrate involved in eliciting oromotor behaviors in response to taste input.

Sequential activation of Snail1 and N-Myc modulates sonic hedgehog-induced transformation of neural cells.

Activation of the Sonic hedgehog (Shh) pathway and increased expression of Gli1 play an important role in proliferation and transformation of granule cell progenitors (GCP) in the developing cerebellum. Medulloblastomas arising from cerebellar GCPs are frequently driven by Shh pathway-activating mutations; however, molecular mechanisms of Shh pathway dysregulation and transformation of neural progenitors remain poorly defined. We report that the transcription factor and oncogene Snail1 (Sna1) is directly induced by Shh pathway activity in GCPs, murine medulloblastomas, and human medulloblastoma cells. Enforced expression of Sna1 was sufficient to induce GCPs and medulloblastoma cell proliferation in the absence of Shh/Gli1 exposure. In addition, enforced expression of Sna1 increased transformation of medulloblastoma cells in vitro and in vivo. Analysis of potential Sna1 targets in neural cells revealed a novel Sna1 target, N-Myc, a transcription factor known to play a role in Shh-mediated GCP proliferation and medulloblastoma formation. We found that Sna1 directly induced transcription of N-Myc in human medulloblastoma cells and that depletion of N-Myc ablated the Sna1-induced proliferation and transformation. Taken together, these results provide further insight into the mechanism of Shh-induced transformation of neural progenitor cells and suggest that induction of Sna1 may serve to amplify the oncogenic potential of Shh pathway activation through N-Myc induction.

Neural stem cell- and Schwann cell-loaded biodegradable polymer scaffolds support axonal regeneration in the transected spinal cord.

Biodegradable polymer scaffolds provide an excellent approach to quantifying critical factors necessary for restoration of function after a transection spinal cord injury. Neural stem cells (NSCs) and Schwann cells (SCs) support axonal regeneration. This study examines the compatibility of NSCs and SCs with the poly-lactic-co-glycolic acid polymer scaffold and quantitatively assesses their potential to promote regeneration after a spinal cord transection injury in rats. NSCs were cultured as neurospheres and characterized by immunostaining for nestin (NSCs), glial fibrillary acidic protein (GFAP) (astrocytes), betaIII-tubulin (immature neurons), oligodendrocyte-4 (immature oligodendrocytes), and myelin oligodendrocyte (mature oligodendrocytes), while SCs were characterized by immunostaining for S-100. Rats with transection injuries received scaffold implants containing NSCs (n=17), SCs (n=17), and no cells (control) (n=8). The degree of axonal regeneration was determined by counting neurofilament-stained axons through the scaffold channels 1 month after transplantation. Serial sectioning through the scaffold channels in NSC- and SC-treated groups revealed the presence of nestin, neurofilament, S-100, and betaIII tubulin-positive cells. GFAP-positive cells were only seen at the spinal cord-scaffold border. There were significantly more axons in the NSC- and SC- treated groups compared to the control group. In conclusion, biodegradable scaffolds with aligned columns seeded with NSCs or SCs facilitate regeneration across the transected spinal cord. Further, these multichannel biodegradable polymer scaffolds effectively serve as platforms for quantitative analysis of axonal regeneration.

Differential gene induction by genetic and ligand-mediated activation of the Sonic hedgehog pathway in neural stem cells.

Sonic hedgehog (Shh), a secreted morphogen and mitogen, is essential for nervous system development and neural stem cell (NSC) self-renewal. As the intracellular signal transduction of Shh in NSCs is largely unknown, we sought to characterize pathway targets using ligand stimulation and genetic models of activation. NSCs haploinsufficient for Patched (Ptc), a receptor repressive to Shh signaling, showed enhanced proliferation of a magnitude similar to Shh-treated wild-type (Wt) NSCs. Analysis of the Gli zinc-finger transcription factors, primary mediators of Shh activity, demonstrated differential induction between models of pathway activation. Gli1 was significantly induced in Wt NSCs exposed to Shh, whereas Gli2 was elevated and Gli1 expression did not change in Ptc(+/-) NSCs. Other Shh targets (Nmyc, Id factors) were induced under both conditions of pathway activation. Interestingly, Shh-treated Ptc(+/-) NSCs induced expression of Gli1 but failed to increase proliferation, suggesting that the NSCs may have reached a physiologic plateau in proliferative capacity. Thus, our data demonstrate that Ptc(+/-) mice have an expanded progenitor cell niche in vivo and that NSCs maintain a cell-intrinsic increase in basal proliferation in vitro that is sustained by a Gli transduction signature distinct from that of exogenous Shh stimulation. Additionally, Ptc(+/-) NSCs maintain tight control over mitosis and do not further augment proliferation in the presence of mitogenic stimulation.