During development, radial glia comprise neurogenic, but at later stages also gliogenic progenitors (Kriegstein and Alvarez-Buylla, 2009). In addition, some radial glial cells give rise to the neural stem cells responsible for adult neurogenesis (Merkle et al., 2004; Kriegstein and Alvarez-Buylla, 2009). Yet little is known about the molecular
determinants that regulate radial glia self-renewal and expansion of the cortex. Using a new antibody raised against Trnp1 I was able to show its expression at protein level in neurogenic niches both during development and in the adult murine brain. The expression of Trnp1 in only a subpopulation of Pax6-positive radial glial cells at mid neurogenesis together with its initial identification in directly neurogenic radial glial cells suggested a specific regulatory role of Trnp1 in neurogenesis. Functional analysis presented in this work identified Trnp1 as a novel regulator of brain development and neocortical expansion. First, to directly examine Trnp1 function a retroviral vector was constructed to overexpress Trnp1. Forced expression in dissociated primary cell cultures derived from the cerebral cortex of E14 mouse embryos resulted in increased proliferation and self-renewal of neural stem cells. This finding was further corroborated with live imaging and single cell tracking which showed an impressive increase of symmetric progenitor division and sustained neurogenesis over a longer time. In utero electroporation to overexpress Trnp1 in the developing cerebral cortex revealed a significant increase of radial glia proliferation in vivo supporting the in vitro findings. In contrast, knock down experiments using short hairpin RNA strongly increased the number of BPs and neurons after three days in vivo. These data show, that high levels of Trnp1 lead to self-renewal of neurogenic radial glial cells, whereas low levels promote increased production of basal progenitors and neuronal output. This result is consistent with our initial identification of Trnp1 in the radial glial subset that does not generate basal progenitors during forebrain development (Pinto et al., 2008).
During brain evolution a remarkable increase in size of the cerebral cortex has occurred especially amongst mammals. One open key question is how expansion of this brain region is regulated at the molecular level and which mechanisms distinguish larger cerebral cortices from smaller ones. Gyrification seems to have developed a long time ago as a blueprint for today’s mammalian brains (Reillo et al., 2011; Borrell and Reillo, 2012; Martínez-Cerdeño et al., 2012). Some species kept the ability to form cortical folds, whereas others lost this ability (Kriegstein et al., 2006; Borrell and Reillo, 2012; Kelava et al., 2012; Martínez-Cerdeño et al., 2012). This hypothesis provokes the question what distinguished that blueprint ancestor from other species, and what factors may have been responsible for such an evolution. In this context it is very tempting to think about factors
that may have developed such a function in the mammalian lineage and are involved in these processes. Trnp1 was initially identified in a screen for directly neurogenic radial glial cells. This sub population is suggested to give rise to neurons directly and to self renew itself trough asymmetric divisions (Miyata et al., 2001; Noctor et al., 2001; Haubensak et al., 2004; Noctor et al., 2004; Pinto and Götz, 2007; Stancik et al., 2010). Acute loss of Trnp1 not only increased the number of BPs but also led to a dramatic local radial expansion of the cortex. This is consistent with previous studies suggesting basal progenitors as the main source of increased neuronal output and expansion (Haubensak et al., 2004; Miyata et al., 2004; Noctor et al., 2004; Kriegstein et al., 2006; Pontious et al., 2008; Sessa et al., 2008; 2010; Hevner and Haydar, 2012). Besides BPs outer radial glial cells (oRGs) lacking an apical process have recently been suggested to be a prerequisite of brain expansion and folding / gyrification of the cortex (Fietz et al., 2010; Hansen et al., 2010; Lui et al., 2011; Molnár, 2011; Reillo et al., 2011). Down regulation of Trnp1 was able to increase the number of such outer radial glial cells in the murine brain. The striking folding of the brain upon Trnp1 knockdown strongly supported this observation and is likely a consequence of both the increase in BP and oRG numbers. In summary, loss of Trnp1 was able to acutely reproduce several hallmarks of larger brain development: 1) strong radial expansion, 2) diverging radial fibers forming a fanned array as has been described for gyrencephalic brains (Smart and McSherry, 1986a; 1986b; Lui et al., 2011; Reillo et al., 2011), 3) increased numbers of oRGs that have been suggested to be a characteristic of larger brains (Fietz et al., 2010; Hansen et al., 2010; Reillo et al., 2011; Wang et al., 2011; Borrell and Reillo, 2012; Martínez-Cerdeño et al., 2012), 4) increased proportion of Tbr2+ basal progenitors. Additionally, a strong increase of the DAPI dense SVZ region in combination with a diffuse Tbr2+ band was found upon down regulation of Trnp1. These observations are indicative of an increased SVZ and somewhat reminiscent of the diffuse band of BPs present in the oSVZ of higher mammalian brains (Martínez- Cerdeño et al., 2012). Altogether, these data suggest that Trnp1 represents a molecular switch that needs to be expressed or “ON” in progenitors in order to increase the progenitor pool and lead to a tangential (lateral) expansion. Accordingly, at the time of strong lateral expansion of forebrain development (i.e. in neuroepithelial cells) Trnp1 is expressed in virtually all progenitors. Later, when the time for increased neuronal
production has come Trnp1 is acutely down regulated in subsets of radial glia to initiate abventricular amplification by basal progenitors and oRGs that then together increase the number of neurons and their guidance structures resulting in radial expansion (see model Figure 38). Therefore, Trnp1 controls progenitor pool expansion and subsequently neuronal expansion. In this context, the timing of Trnp1 down regulation may determine the extent of radial expansion. Acute loss of Trnp1 immediately increases neuronal output at the expense of remaining apical radial glial cells. Hence, acute loss of Trnp1 will presumably lead to an acute radial expansion. This suggests, that regulation of Trnp1 expression may be involved in radial expansion and gyrification to accommodate the additional neuron numbers in larger brains. The process of oRG generation in the murine brain is endogenously very inefficient and slow as oRGs are found only sparsely (Shitamukai et al., 2011; Wang et al., 2011). The level of Trnp1 seems to play a critical role in this process and knock down of Trnp1 released the inhibition of oRG production. The loss of apical radial glia upon Trnp1 down regulation seems to be compensated by the increase in oRG numbers that have been shown to have a higher proliferation coefficient than BPs (Lui et al., 2011).
In summary, using both clonal analysis in vitro and in utero electroporation in vivo Trnp1 was found to be a master regulator of radial glial fate. So far, Trnp1 represents the first factor identified to regulate the generation of both oRGs and BPs at the same time. In strong contrast to Volpe et al, Trnp1 was not simply increasing proliferation but it does rather represent a crucial regulator of cellular fate and neurogenesis. Additionally, Trnp1 also represents the first factor capable to generate folding in an otherwise lissencephalic brain recapitulating all the hallmarks of higher mammalian gyrencephalic brain development. In this context, it should be mentioned that a transgenic mouse line expressing a stabilized form of beta catenin has previously been reported to show folding of the neocortex (Chenn and Walsh, 2002). However, in strong contrast to the observed local expansion and folding upon Trnp1 knock down, the beta catenin induced folding did not resemble folding of higher mammalian gyrencephalic brain, but rather represents a secondary folding due to lateral expansion of the progenitor pool. Importantly, the folding observed by Chenn et al. included folding and expansion of the ventricular surface and did not fulfill characteristics of radial expansion. In contrast, down-regulation of Trnp1
resulted in a local radial expansion and folding of the cortical plate without increasing the ventricular surface therby reflecting the situation in gyrencephalic brains. Together with the above mentioned characteristics such as the appearance of oRG cells, timed and local manipulation of Trnp1 was able to recapitulate central features of gyrencephalic brains.
A A’
B’ B
Basal progenitor Outer radial glial cell
Radial glial cell with low levels of Trnp1 expression
Differentiating neuron
Radial glial cell with high levels of Trnp1 expression Migrating neuroblast Knock down of Trnp1 Overexpression of Trnp1 Tangential expansion Radial expansion
Figure 38: Model of Trnp1 action during mammalian cortical brain development
Illustration of the mode of Trnp1 action in neural stem cells during cerebral cortical development. (A) High levels of Trnp1 in radial glial cells lead to proliferation and self renewal of radial glia with either symmetric divisions to generate two radial glial cells or asymmetric division allowing direct neurogenesis to occur at the same time. (A’) High levels of Trnp1 enlarge the pool of stem cells leading to tangential expansion. (B) In contrast to that, low levels of Trnp1 lead to the generation of basal progenitor cells that subsequently amplify the neuronal output. At the same time, low levels of Trnp1 also provoke generation of outer radial glia (oRG) that lost their apical process and serve to amplify the neuronal output but also are essential for folding of an expanding cortex. (B’) Both the high number of basal progenitors and the generation of oRGs together lead to a radial expansion of the cortex and ultimately result in folding of the brain as naturally occurring in higher mammalian species.