Munekazu Komada, PhD, Division of Cell Biology and Neuroscience, Department of Morphological and Physiological Sciences, University of Fukui, 23 Matsuokashimoaizuki, Eiheiji-cho, Fukui 910-1193, Japan. Email: firstname.lastname@example.org
Sonic hedgehog (Shh) acts as a morphogen in normal development of various vertebrate tissues and organs. Shh signaling is essential for patterning and cell-fate specification, particularly in the central nervous system. Shh signaling plays different roles depending on its concentration, area, and timing of exposure. During the development of the neocortex, a low level of Shh is expressed in the neural stem/progenitor cells as well as in mature neurons in the dorsal telencephalon. Shh signaling in neocortex development has been shown to regulate cell cycle kinetics of radial glial cells and intermediate progenitor cells, thereby maintaining the proliferation, survival and differentiation of neurons in the neocortex. During the development of the telencephalon, endogenous Shh signaling is involved in the transition of slow-cycling neural stem cells to fast-cycling neural progenitor cells. It seems that high-level Shh signaling in the ventral telencephalon is essential for ventral specification, while low-level Shh signaling in the dorsal telencephalon plays important roles in the fine-tuning of cell cycle kinetics. The Shh levels and multiple functions of Shh signaling are important for proper corticogenesis in the developing brain. The present paper discusses the roles of Shh signaling in the proliferation and differentiation of neural stem/progenitor cells.
Sonic hedgehog (Shh) is a secreted protein that acts as a morphogen in the normal development of various tissues and organs in vertebrates, such as the limb, neural tube, heart, and cerebellum (Chiang et al. 1996; Dahmane et al. 2001; Ruiz i Altaba et al. 2002). During the development of the mammalian central nervous system (CNS), Shh plays important roles as a morphogenic factor in ventral patterning, proliferation, differentiation and survival of neural stem/progenitor cells along the neuraxis, including the telencephalon (Roelink et al. 1995; Ruiz i Altaba et al. 1995; Agarwala et al. 2001). The pivotal roles of Shh signaling in the development of CNS have been elucidated by the study of Shh knock out (KO) mouse embryos. When the Shh gene is knocked out in mice, the ventral structures of the CNS, midline craniofacial structures, and other organs are severely affected in mutant embryos (Chiang et al. 1996). In the human, SHH mutations have been shown to result in serious midline anomalies of the brain and face, such as holoprosencephaly (HPE). HPE and related anomalies in the human are consistent with the phenotypes of Shh KO mice and Shh gene has been shown to be one of the major genes responsible for human HPE. On the other hand, recent studies have revealed that Shh may be involved in the development of the dorsal brain, including the neocortex. In the present paper, the functions of Shh signaling in the development and differentiation of the dorsal brain are discussed, with special reference to the morphogenesis of the neocortex (dorsal telencephalon).
DEVELOPMENT OF THE NEOCORTEX
The telencephalon is developed from the most anterior part of the neural tube. Neural epithelial cells in the ventricular surface of the neural tube proliferate to expand the neural tube. During the development of the dorsal telencephalon, neural stem/progenitor cells sequentially pass through the phases of proliferation and differentiation (neurogenesis and gliogenesis). Neural stem cells having a radial fiber in the ventricular zone (VZ) are called radial glial cells (RGCs). During the early phase of differentiation, RGCs proliferate by symmetric division and produce two RGCs, but later they undergo asymmetric cell division to simultaneously self-renew and generate more differentiated cells (Gotz and Huttner 2005). Neuronal progenitor cells (Intermediate progenitor cells; IPCs) in the subventricular zone (SVZ), which are a kind of daughter cell of RGCs, migrate into the SVZ to undergo further asymmetric cell division to produce a neuron and an RGC or an IPC and an RGC (Haubensak et al. 2004; Noctor et al. 2004; Miyata et al. 2010). While IPCs usually undergo terminal asymmetric division to produce a neuron, the division of IPCs may be symmetric to achieve self-renewal, depending on their situation (Noctor et al. 2004; Miyata et al. 2010). IPCs are assumed to be involved in the development of the neocortex during mammalian evolution, because IPCs have a high ability of self-renewal and neurogenesis during the neocortical development (Molnar et al. 2011).
During normal development of the mouse neocortex, RGCs and IPCs proliferate in the proliferative zone (VZ/SVZ), and eventually differentiate into neurons. These neurons migrate radially out of the VZ/SVZ and accumulate below the brain surface to form the cortical plate (CP). The neurons in the CP are generated in an inside-out gradient and produce a six-layer structure (Molyneaux et al. 2007). The major cortical neurons are the pyramidal neurons (derived from the dorsal telencephalon) and GABAergic interneurons (derived from the ventral telencephalon). These neurons produce Shh protein in the dorsal telencephalon and play important roles in both the development and function of the neocortex (Fuccillo et al. 2004; Xu et al. 2005; Gulacsi and Anderson 2006).
Shh signaling in the ventral telencephalon
Previous studies have shown that Shh signaling plays pivotal roles in the forebrain development throughout the pre- and postnatal stages. The ventral regions of telencephalon are severely affected in Shh KO mouse embryos (Chiang et al. 1996). In the early stage of embryonic development, Shh is required for the specification of the cells in the basal and floor plates and motor neurons in the spinal cord (Marti et al. 1995; Roelink et al. 1995; Chiang et al. 1996). In the middle stage of embryonic development, Shh expresses in the medial ganglionic eminence (MGE) of the ventral telencephalon and controls the expression of transcriptional factors, Nkx2.1 and Gsh2, which are involved in the ventral patterning of the telencephalon (Corbin et al. 2003). In addition, it seems that Shh signaling regulates the proliferation and differentiation of oligodendrocytes and GABAergic interneurons by means of these transcriptional factors during the development of the ventral telencephalon (Corbin et al. 2003).
Shh signaling in the development of the dorsal telencephalon
Recent studies have shown that Shh signaling is involved in the development of not only the ventral region but also the dorsal part of the telencephalon. In the dorsal telencephalon of perinatal mice, Shh mRNA was detectable only by reverse transcription-polymerase chain reaction in the dorsal telencephalon from E14.5 to P3 (Dahmane et al. 2001; Palma and Ruiz i Altaba 2004). However, in our previous study, we detected Shh protein expression in the developing dorsal telencephalon by immunostaining methods (Komada et al. 2008). Shh-positive cells included RGCs, IPCs and mature neurons (projection neurons, Cajal–Retzius neurons, and GABAergic interneurons) in the developing dorsal telencephalon.
In the dorsal telencephalon of Smoc/–;Foxg1Cre and Smon/c;NestinCre conditional KO (cKO) mice, in which Smo genes were specifically knocked out in the developing telencephalon, the cortical thickness was reduced due to an increase in apoptosis and a decrease in the number of interneurons (Machold et al. 2003; Fuccillo et al. 2004; Xu et al. 2005). On the contrary, the neocortex became hyperplastic when Shh signaling was promoted. Lien et al. (2006) reported that in αE-cateninloxP/loxP/Nestin-Cre+/− mice, the disruption of alphaE-catenin connecting cell density-dependent adherens junction induced abnormal activation of hedgehog signaling in the dorsal telencephalon, resulting in shortened cell cycle, decreased apoptosis and hyperplastic cortex (Lien et al. 2006). In addition, a recent study using in utero electroporation revealed that Shh signaling affects the transition of RGCs to IPCs and activates the proliferation of IPCs in the dorsal telencephalon late in gestation (Shikata et al. 2010). Furthermore, when Shh and Smo genes were specifically knocked out in the developing dorsal telencephalon of Emx1-Cre knock-in mice the dorsal telencephalon of Emx1Cre;Shhfl/− and Emx1Cre;Smofl/− mouse fetuses was significantly smaller at E18.5 than in wild-type fetuses, while the morphology of the ventral telencephalon was not significantly altered (Iwasato et al. 2004; Komada et al. 2008). These studies indicate that Shh signaling regulates the growth and development of the dorsal telencephalon and coordinates the thickness of the neocortex.
Cell proliferation and survival in the embryonic and fetal telencephalon
For normal morphogenesis of the neocortex, cell proliferation and survival need to be appropriately coordinated. When proliferation, neurogenesis and cell death are dysregulated in the dorsal telencephalon, macroencephaly and microencephaly are produced (Xu et al. 2005; Lien et al. 2006). We revealed by immunostaining and BrdU incorporation studies that the proliferation of RGCs and IPCs in the dorsal telencephalon was significantly decreased in Emx1Cre;Shhfl/− and Emx1Cre;Smofl/− mice (Komada et al. 2008). In particular, defective Shh signaling decreased the number of IPC (TBR2-positive neural progenitor cells) in the dorsal telencephalon of Emx1Cre;Shhfl/−, Emx1Cre;Smofl/− mice (Komada et al. 2008) and Shh null mutant (Shikata et al. 2010). In addition, when Shh signaling was upregulated during cortical neurogenesis the thickness and size of the neocortex were significantly increased (Lien et al. 2006; Komada et al. 2008; Shikata et al. 2010) (Fig 1). These data suggest that Shh signaling promotes the proliferation of neural stem/progenitor cells in the dorsal telencephalon during development.
Another important mechanism to control the brain size is the regulation of apoptosis of RGCs, IPCs and mature neurons in the fetal brain. In this regard, it is interesting to note that when both caspase-3 and caspase-9 (essential factors for cell death) are knocked out, apoptosis was decreased in the brain, resulting in the excessive cell number and deformed cerebral hyperplasia (Tang 2006). On the contrary, apoptotic cells increased in the dorsal telencephalon in Emx1Cre;Shhfl/− and Emx1Cre;Smofl/− (Komada et al. 2008) and Smoc/–;Foxg1Cre mice in which Shh signaling was selectively knocked out (Fuccillo et al. 2004). In addition, apoptosis was reduced in the dorsal telencephalon of αE-cateninloxP/loxP/Nestin-Cre+/- mice where Shh signaling was abnormally activated (Lien et al. 2006). Thus, it is likely that Shh signaling controls the brain size partly by controlling the proliferation and apoptotic death of neural stem/progenitor cells and neurons.
In the developing dorsal telencephalon, Shh signaling seems to coordinate the cell cycle kinetics and the transition of RGCs to IPCs and IPCs to cortical neurons (Komada et al. 2008; Shikata et al., 2010) (Fig 2). In Emx1Cre;Smofl/− mice where Shh signaling was knocked out in RGCs and IPCs, cell division was slower than that in wild-type mice and the exit from the cell cycle to become non-dividing neurons was impaired (Komada et al. 2008) (Fig 2). In αE-cateninloxP/loxP/Nestin-Cre+/–, the shortening of the cell cycle of neural stem/progenitor cells resulted in hypoplasia of the neocortex (Lien et al. 2006). Shikata et al. (2010) upregulated Shh signaling using in utero electroporation and enhanced the transition of RGCs to IPCs in the dorsal telencephalon (Shikata et al. 2010). Shh signaling has been shown to affect various checkpoints of the cell cycle by controlling activities of cyclins and cyclin-dependent kinases (Ruiz i Altaba et al. 2002). Thus, Shh signaling controls cell cycle kinetics by regulating cell cycle regulators. It seems that in the development of neocortex, fine-tuning of the cell cycle by Shh signaling is essential for proper proliferation and survival of RGCs and IPCs.
Neuronal positioning and lamination in the neocortex
Neuronal positioning and lamination in the brain are closely related to the cell cycle exit of neural progenitor cells (Caviness et al. 2009). In the normal developing brain, postmitotic neurons migrate out of the proliferative zones and accumulate to form the CP (O'Leary and Koester 1993). In order to examine the relationship between the timing of neurogenesis and migration and the distribution of neurons and layer identities of the neocortex, we performed a birthdate analysis and revealed that in wild-type embryos, early-born neurons are settled down mainly in the deeper layer and late-born neurons migrate to the more superficial layers of the CP (Fig 3), thereby inside-out pattern of the CP is formed. It was also noteworthy that neuronal positioning pattern was severely impaired in Emx1Cre;Smofl/− mice. In their brain, early-born neurons were scattered throughout the dorsal telencephalon and late-born neurons failed to migrate to the superficial layer of the CP (Komada et al. 2008) (Fig 3). In addition, in Emx1Cre;Smofl/− mice in which Shh signaling was lacking, the expression of layer specific markers in the neocortex were disrupted (Komada et al. 2008). Thus, the neuronal distribution and the formation of laminar structure in the neocortex are regulated by cell cycle kinetics controlled by Shh signaling and the disruption of the neuronal distribution affects the layer specification in the neocortex.
Interaction between Shh and Wnt signalings in telencephalic development
It has been shown that Wnt signaling is required for the development of the telencephalon and Wnt and Shh signaling control the differentiation of the dorsal versus ventral telencephalic neurons (glutamatagic neuron versus GABAergic neurons) from progenitor cells by modulating Gli3 (active or repressive form) (Li et al. 2009). Thus, the proper coordination of Wnt and Shh signaling through Gli3 induction may be important for the dorso-ventral patterning in the developing telencephalon.
Recent studies have revealed that β-catenin-mediated Wnt signaling is involved in regulating the proliferation and differentiation of neural progenitor cells in the dorsal telencephalon. In addition, if Wnt signaling is blocked in the MGE, the proliferation of neural progenitor cells decreases and the growth of the MGE is impaired (Gulacsi and Anderson 2008). Thus, Wnt signaling regulates the growth of the ventral telencephalon. In the neural tube, Shh and Wnt signalings mediate the growth gradient that coordinates the increased growth of ventral and dorsal regions (Ulloa and Marti 2010). Shh and Wnt signalings show similar characteristics in the telencephalon. Shh and Wnt signalings are initially involved in fate determination in the ventral and dorsal telencephalon, respectively, while later in neurogenesis, they regulate the proliferation and differentiation of the opposing fields (dorsal and ventral field, respectively).
The multiple roles of Shh signaling during the development of CNS depend on the region, timing, and concentration of Shh expression (Fig. 4). The Shh expression level in the dorsal telencephalon is extremely low as compared with that in the ventral telencephalon (MGE). Endogenous ‘high level’Shh signaling is expressed in the MGE and the signaling is important for ventral patterning and differentiation of GABAergic interneurons. Endogenous ‘low level’Shh signaling in the dorsal telencephalon also plays important roles in the proliferation, differentiation, and positioning of cortical neurons through fine-tuning of cell cycle kinetics. Therefore, Shh signaling plays multiple roles during the development of the telencephalon, depending on the region, concentration and the timing of its expression (Komada et al. 2008; Shikata et al. 2010).
Shh signaling is involved in the proliferation of RGCs and IPCs during the development of the neocortex. In addition, Shh signaling regulates the cell cycle of RGCs and IPCs in the dorsal telencephalon and thereby controls the proliferation, cell death, transition from RGCs to IPCs and neurogenesis (Fig. 4). It seems that Shh signaling is important for proper corticogenesis and works in coordination with Wnt signaling to assume different roles in the embryonic dorsal telencephalon, depending on its concentration and the development stage.
The author is grateful to Drs Kohei Shiota and Makoto Ishibashi for their continued support and collaboration and to Dr Makoto Sato for critical comments on the manuscript. The original works cited in this paper were carried out in the Department of Anatomy and Developmental Biology, Kyoto University Graduate School of Medicine. Figures 1–3 were adapted with permission from Komada et al. (2008).
Grant information: Grant sponsor: Japanese Ministry of Education, Culture, Sports, Science and Technology; Grant number: 15689004, 16015264, 18590168, 22790186).