Sonic Hedgehog and Notch Signaling Can Cooperate to Regulate Neurogenic Divisions of Neocortical Progenitors Richa K. Dave1, Tammy Ellis1, Melissa C. Toumpas1, Jonathan P. Robson1, Elaine Julian 1 , Christelle Adolphe1, Perry F. Bartlett2, Helen M. Cooper2,3, Brent A. Reynolds2¤, Brandon J. Wainwright1* 1 The Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia, 2 Queensland Brain Institute, The University of Queensland, Brisbane, Queensland, Australia, 3 School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia Abstract Background: Hedgehog (Hh) signaling is crucial for the generation and maintenance of both embryonic and adult stem cells, thereby regulating development and tissue homeostasis. In the developing neocortex, Sonic Hedgehog (Shh) regulates neural progenitor cell proliferation. During neurogenesis, radial glial cells of the ventricular zone (VZ) are the predominant neocortical progenitors that generate neurons through both symmetric and asymmetric divisions. Despite its importance, relatively little is known of the molecular pathways that control the switch from symmetric proliferative to differentiative/neurogenic divisions in neural progenitors. Principal Findings: Here, we report that conditional inactivation of Patched1, a negative regulator of the Shh pathway, in Nestin positive neural progenitors of the neocortex leads to lamination defects due to improper corticogenesis and an increase in the number of symmetric proliferative divisions of the radial glial cells. Hedgehog-activated VZ progenitor cells demonstrated a concomitant upregulation of Hes1 and Blbp, downstream targets of Notch signaling. The Notch signaling pathway plays a pivotal role in the maintenance of stem/progenitor cells and the regulation of glial versus neuronal identity. To study the effect of Notch signaling on Hh-activated neural progenitors, we inactivated both Patched1 and Rbpj, a transcriptional mediator of Notch signaling, in Nestin positive cells of the neocortex. Conclusions: Our data indicate that by mid neurogenesis (embryonic day 14.5), attenuation of Notch signaling reverses the effect of Patched1 deletion on neurogenesis by restoring the balance between symmetric proliferative and neurogenic divisions. Hence, our results demonstrate that correct corticogenesis is an outcome of the interplay between the Hh and Notch signaling pathways. Citation: Dave RK, Ellis T, Toumpas MC, Robson JP, Julian E, et al. (2011) Sonic Hedgehog and Notch Signaling Can Cooperate to Regulate Neurogenic Divisions of Neocortical Progenitors. PLoS ONE 6(2): e14680. doi:10.1371/journal.pone.0014680 Editor: Mai Har Sham, The University of Hong Kong, China Received May 13, 2010; Accepted January 3, 2011; Published February 17, 2011 Copyright: 2011 Dave et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Australian National Health and Medical Research Council and the ARC Special Research Centre for Functional and Applied Genomics. Confocal microscopy was performed at the Australian Cancer Research Foundation (ACRF)/Institute for Molecular Bioscience Dynamic Imaging Facility for Cancer Biology, which was established with the support of the ACRF. R. Dave is supported by the Queensland Cancer Fund. T. Ellis is supported by The John Trivett Foundation Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: b.wainwright@imb.uq.edu.au ¤ Current address: Evelyn F. and William L. McKnight Brain Institute, University of Florida, Gainesville, Florida, United States of America Introduction The development of the mammalian neocortex requires the orchestration of numerous processes in a spatiotemporal manner resulting in the precise six-layered structure. Neurons and radial glia are the major cell types that constitute the vertebrate nervous system and are derived from the neuroepithelial cells, the primary neural progenitors [1]. After embryonic day (E)10, neuroepithelial cells give rise to radial glial cells, which then constitute the predominant progenitor population in the neocortex [2]. These radial glial cells serve both as neural precursors and migratory scaffolds. At the peak of neurogenesis, radial glial cells can undergo either symmetric proliferative divisions to maintain their own pool or can differentiate into either a basal progenitor (BP) that populates the newly formed subventricular zone (SVZ), or a neuron [3,4,5,6]. These post-mitotic neurons subsequently migrate up the radial glial cells to form the cortical plate (CP). Corticogenesis thus requires the co-ordination of signaling pathways that provide mitogenic signals, positional information, migratory cues, differentiation signals, and a requirement for the precise regulation of symmetric/asymmetric division. Although some of these critical signaling pathways have been defined, in many examples we still lack a detailed understanding of the precise consequences of the interaction between them and the factors that influence the number and type of divisions a neocortical progenitor cell undergoes. The Hedgehog signaling pathway plays a key role in the ventral patterning of the entire neuraxis, powerfully illustrated by the PLoS ONE | www.plosone.org 1 February 2011 | Volume 6 | Issue 2 | e14680