FMRP regulates the transition from radial glial cells to intermediate progenitor cells during neocortical development.

“The RGC-to-IPC transition, a key step in neuronal production from RGCs, may underlie the evolutionary expansion of the cerebral cortex (Fish et al., 2008; Kriegstein and Alvarez-Buylla, 2009).”

“To overcome this problem, we used Lifeact-EGFP (Riedl et al., 2008, 2010), a fusion protein comprised of an F-actin binding domain and EGFP, to visualize the ring-like F-actin structure in transfected RGCs (Fig.”

“The I304N mutation impairs the association of FMRP with RNA (Bassell and Warren, 2008) and renders the protein a null mutant (Zang et al., 2009).”

“Moreover, potential function of FMRP in RGCs may be masked in Fmr1 knock-out embryos by compensation, which often occurs in constitutive knock-out mice (Deuel et al., 2006; Koizumi et al., 2006; Fasano et al., 2007; Satyanarayana and Kaldis, 2009).”

“Immediately following mCherry electroporation, EGFP should label cells with reduced levels of FMRP, because FMRP shRNA was coelectroporated with pCAX-EGFP 2 d before, and mCherry should label RGCs because in utero electroporation leads to specific transfection of RGCs (Gal et al., 2006).”

“To overcome this problem, we used Lifeact-EGFP (Riedl et al., 2008, 2010), a fusion protein comprised of an F-actin binding domain and EGFP, to visualize the ring-like F-actin structure in transfected RGCs (Fig.”

“These three proteins have similar domain structures and interact with each other (Zhang et al., 1995).”

“FMRP maintains the RGC pool by regulating the actin cytoskeleton Previous studies indicate that FMRP may regulate neuronal function by an actin-dependent mechanism (Schenck et al., 2003; Reeve et al., 2005).”

“Moreover, potential function of FMRP in RGCs may be masked in Fmr1 knock-out embryos by compensation, which often occurs in constitutive knock-out mice (Deuel et al., 2006; Koizumi et al., 2006; Fasano et al., 2007; Satyanarayana and Kaldis, 2009).”

“Thus, the transition from RGCs to IPCs plays a key role in determining the total number of neurons produced from RGCs and may underlie the evolutionary expansion of the cerebral cortex (Fish et al., 2008; Kriegstein and Alvarez-Buylla, 2009).”

“…have shown that functional compensation commonly occurs during development when the gene is constitutively deleted but not when the protein is acutely lost or inhibited (Deuel et al., 2006; Koizumi et al., 2006; Fasano et al., 2007; Satyanarayana and cortical neurogenesis than at later stages.”

“Several actin-dependent cellular mechanisms may control the cell fate decision of RGC divisions, including the regulation of apical polarity, interkinetic nuclear migration, and cleavage plane (Farkas and Huttner, 2008; Fish et al., 2008; Kriegstein and Alvarez-Buylla, 2009).”

“In particular, FMRP was recently shown to play an important role in the proliferation of neural stem cells in the Drosophila larval brain (Callan et al., 2010) and in the adult mouse brain (Luo et al., 2010).”

“Nonetheless, we found that the H119E mutation, which specifically impairs the actin-binding activity of Pfn1 (Suetsugu et al., 1998), abolished the rescue of RGC depletion by Pfn1 (Fig.”

“For instance, the genetic interaction between dFmr1 and dRac1 (Lee et al., 2003; Schenck et al., 2003) and that between dFmr1 and aPKC (Zarnescu et al., 2005) are either antagonistic or synergistic, depending on the cellular contexts.”

“In neocortical neural stem cells, F-actin forms a distinct ring-like structure at the apical endfeet (Chenn et al., 1998).”

“Magnetic resonance imaging analysis of FXS patients has revealed widespread bidirectional brain volume changes (Gothelf et al., 2008).”

“This explanation would be consistent with the previous observation that FMRP deficiency leads to increased neuronal production in cultured neurospheres (Castrén et al., 2005).”

“In Drosophila, dFmr1 (ortholog of FMRP) genetically interacts with profilin (Reeve et al., 2005), a core actin-regulatory protein that promotes F-actin formation (Birbach, 2008).”

“The I304N mutation impairs the association of FMRP with RNA (Bassell and Warren, 2008) and renders the protein a null mutant (Zang et al., 2009).”

“Furthermore, analysis of skeletal and cardiac muscles in Fxr1 knock-out mouse embryos has revealed actin defects in these tissues (Mientjes et al., 2004).”

“Behavioral analysis of Fmr1 Fxr2 double knock-out mice suggests that the loss of FMRP may be partially compensated for by FXR2 (Spencer et al., 2006), although the mechanism of compensation remains unclear.”

“5 (Miyama et al., 1997).”

“In Drosophila, dFmr1 (ortholog of FMRP) genetically interacts with profilin (Reeve et al., 2005), a core actin-regulatory protein that promotes F-actin formation (Birbach, 2008).”

“Although a previous study (Tervonen et al., 2009) observed normal expression of cytoplasmic markers of RGCs in Fmr1 knock-out mouse embryos during late neocortical neurogenesis, a possible change in RGC numbers during earlier stages of neocortical neurogenesis cannot be ruled out.”

“In particular, FMRP was recently shown to play an important role in the proliferation of neural stem cells in the Drosophila larval brain (Callan et al., 2010) and in the adult mouse brain (Luo et al., 2010).”

“, 2003) and that between dFmr1 and aPKC (Zarnescu et al., 2005) are either antagonistic or synergistic, depending on the cellular contexts.”

“The in utero electroporation procedure was performed as described by Walantus et al. (2007) with modifications.”

“…loss of Fmr1 has been associated with abnormalities in cortical development and dendritic spine formation, which may contribute to aberrant learning and behavior in fragile X syndrome (Castrén et al., 2005; La Fata et al., 2014; Saffary and Xie, 2011; Shen et al., 2019; Tervonen et al., 2009).”

“In utero electroporation was performed as described previously (Saffary and Xie, 2011).”

“…was found to function in neuronal migration, differentiation and dendritic spine maturation (Hinton et al., 1991; Irwin et al., 2001; Saffary and Xie, 2011; Telias et al., 2013; La Fata et al., 2014; Khalfallah et al., 2017), it seems therefore possible that the microsatellite of FMR1 might…”

“…et al. 2011; Chen et al. 2012), but their function in stem cell 1362 GENES & DEVELOPMENT maintenance has been demonstrated in the germline of only a few invertebrate species such as Drosophila, Caenorhabditis elegans, and the planarian Dugesia japonica (Parisi and Lin 2000; Salvetti et al. 2005).”

“Loss-of-functions of FMR protein (FMRP) showed: (i) a switch from AP to BP fate, indicating the depletion of aRGCs pool (Saffary and Xie, 2011); (ii) defects in neuronal positioning due to the misregulation of Frontiers in Cellular Neuroscience | www.frontiersin.org 7 December 2017 | Volume 11 |…”

“P0), consistent with the critical role of FMRP in brain development (49,58), followed by what appears to be a general pattern of decline with age.”

“…abnormalities in the early development in FXS, including abnormal differentiation of neural progenitor cells (NPCs) and impaired migration of newly born neurons (Castrén et al., 2005; Tervonen et al., 2009; Callan and Zarnescu, 2011; Saffary and Zhigang, 2011; Sheridan et al., 2011; Castren, 2012).”

“Moreover, studies have shown that neuronal progenitor cells of Fmr1 knockout mice exhibit aberrant migration and differentiation (Castren et al., 2005; Tervonen et al., 2009; Saffary and Xie, 2011).”

“The adhesion molecule PCDH9 and cytoskeletal proteins PFN1 and TMSB10, molecules involved in the balance of NSC quiescence and activation, were also specifically expressed in these RPCs (Morizur et al., 2018; Saffary and Xie, 2011) (Figure 4E).”

“A knockdown of Fmr1 and Fmr1 knock-out mice resulted in a reduction in the size of the Pax6+ NSC pool due to an NSCto-IPC cell fate change [110].”

“Altered differentiation of FXS iPSC-derived neural progenitors to glutamatergic lineages is consistent with impaired maturation of glutamatergic neurons and their signaling identified during the development of the Fmr1-KO mouse brain (1, 13, 14).”

“Some mRNA targets of FMRP are known to mediate cell proliferation and neurogenesis (Luo et al., 2010), and FMRP deficiency in murine neural stem cells disrupts translation and neuronal differentiation (Liu et al., 2018; Saffary and Xie, 2011).”

“FMRP expression is also developmentally regulated in both humans (Abitbol et al., 1993) and mice (Saffary and Xie, 2011), with expression starting at early embryonic stages, peaking during early post-natal developmental critical periods, but remaining at sustained levels throughout adulthood (Till,…”

“In addition, via direct linkage to cytoskeletal components integrin and cadherin signaling can also lead to modulation of a variety of actin-regulated and other mechanotransductory pathways [140–143].”

“However, oRG will self renew and give rise to neural progenies and IPCs.”

“It was first posited that FMRP interacts with cytoskeleton proteins in RG (Saffary and Xie, 2011).”

“A reduced pool of radial glia and an increased number of Tbr2-positive basal progenitors are shown in the embryonic cortex of Fmr1-KO mice (Castren et al., 2005; Tervonen, et al., 2009; Saffary and Xie, 2011).”

“Disruption of Notch or Fragile-X mental retardation protein signaling promotes the transition of RGCs to IPCs (Mizutani et al., 2007; Saffary and Xie, 2011; Yoon et al., 2008).”

“DISCUSSION Studies have begun to reveal the factors that drive the progression of radial glia into IPCs during development (Farkas et al., 2008; Sessa et al., 2008; Saffary and Xie, 2011).”

“For example, FMRP is thought to play a crucial role in maintenance of radial glial cells (RGCs) in the neocortex during early development (53).”

“…the previously mentioned studies on FMRP’s role in neurogenesis were performed using embryonic or early postnatal day brains (Castren et al., 2005; Saffary and Xie, 2011; Tervonen et al., 2009), Luo et al. set out to test the role of FMRP in adult neural progenitors, particularly within the…”

“Loss of function approaches have shown that in the developing brain, FMRP inhibits the transition from a stem cell-like to an intermediate progenitor fate (Saffary and Xie, 2011).”

“…in Drosophila indicate a negative role of FMRP in regulating profilin expression, studies in mice came to opposite conclusions: overexpression of PFN1 in radial glial cells largely rescues developmental defects during corticogenesis in FMRP-depleted mice (Figure 6.5; Saffary and Xie, 2011).”

“Cell survival was unaffected in Drosophila dFmr1 mutant neuroblast cultures and following acute FMRP knockdown in vivo in embryonic mouse cortex (Callan et al., 2010; Saffary and Xie, 2011).”

“Some RBPs, such as FMRP, may dually function as both translational activators and repressors to influence neurogenesis, depending upon the species [54,55].”

“35 Fragile X mental retardation protein (FMRP) is a 36 mRNA binding protein thought to be important for 37 normal neuronal development [6, 7].”

“Additionally, overexpression of Cyclin D2 leads to an increase in the number of neural progenitor cells, thus confirming its role in neurogenesis (Saffary and Xie, 2011; Tsunekawa et al., 2012).”

“A similar role for the endosome pathway has been described in fungi, suggesting that this mechanism may be evolutionarily conserved.60,61 Indeed, it is possible that endosome and vesicle dependent translation hot spots could occur in mammalian RGCs. Mitochondria have been observed in RGC basal structures, but roles fo…”

“Another study reported that deletion of Fmr1 is associated with abnormalities in cortical development and dendritic spine formation, which leads to abnormal learning and behavior in fragile X syndrome (Castrén et al., 2005; Saffary and Xie, 2011; La Fata et al., 2014).”

“Second, the traditional ASD-linked genes/pathways (Wnt, PTEN, TSC1/TSC2, FMR1) also impinge on NSC self-renewal and differentiation during development (18-21), although these activities in the context of NSC biology are largely ignored in contemporary discussions of mechanisms of ASD”

“While many researchers reported the altered neural stem cell proliferation and differentiation in embryonic and adult brains of Fmr1 KO mice, the mechanistic studies how FMRP modulate stem cell proliferation and differentiation are far from exhaustive (Castren et al. 2005; Tervonen et al. 2009; Saffary and Xie 2011).”

“1F and G), which might not be owing to the effects of FMRP deficiency on extended trailing processes/axon formation during brain development (22,23).”

“…Caille, Lim, GarciaVerdugo, & Alvarez-Buylla, 1999; Gotz, Sirko, Beckers, & Irmler, 2015; Huang & Tan, 2015; Merkle, Tramontin, Garcia-Verdugo, & AlvarezBuylla, 2004) and pathological conditions (Duan et al., 2015; Mo et al., 2018; Pan, Mao, & Sun, 2017; Saffary & Xie, 2011; Shen et al., 2016).”

“…and promoting radial migration, dendrite and dendritic spine development, circuit function, and plasticity (Cunningham et al., 2011; De Rubeis et al., 2013; Goncalves et al., 2013; Hays et al., 2011; La Fata et al., 2014; Nagaoka et al., 2016; Saffary & Xie, 2011; Tervonen et al., 2009).”

“…shown to alter cortical plate development by affecting both the transition from radial glial cells to intermediate precursor cells and the differentiation of excitatory glutamatergic cells (Tervonen et al., 2009; Saffary and Xie, 2011), offering further support for the role of FMRP in neurogenesis.”

“When FMRP was down-regulated, the number of radial glia cells was depleted by the increasing rate of this transition [182].”

“5–17.5 In vivo Promoted a shift from neocortical RGC to IPC (Saffary and Xie 2011) FXS Human fetuses (18 w) In vitro Increased neurons and reduced glia cells from hNSCs (Castren et al. 2005) FXS Human fetuses (14 w) In vitro No differences in proliferation and neuronal differentiation of hNSCs (Bhattacharyya et al. 20…”

“While the importance of FMRP during development has been well established, a potential role for FMRP in mediating alterations in translation in response to chronic EtOH exposure is not known (Bonaccorso et al., 2015; Fernandez et al., 2013; Saffary and Xie, 2011).”

“CP-AMPARs provide an important route for Ca2+ entry into progenitors, and changes of Ca2+ signaling can contribute to altered fate determination, differentiation, and migration of FXS neural progenitors (Castren et al., 2005; Tervonen et al., 2009; Saffary and Xie, 2011; Sheridan et al., 2011; Liu et al., 2012; Doers…”

“FMRP is also present at high levels in radial glial cells (RGCs) of the embryonic mouse neocortex, where it controls mRNAs transport and localization (Pilaz et al., 2016).”

“5 showed a severe depletion in the total pool of RGCs (Saffary and Xie 2011).”

“Saffary and Xie (2011) recently showed that FMR1 utilizes an actin-dependent mechanism to control the ratio of radial glia (RG) and intermediate progenitor cells (IPs) during neurogenesis, suggesting that the observed increase in neurons is due to a shift in the relative ratio of RG to IPs during…”

“Interestingly, profilin1 acts downstream of the fragile X mental retardation protein state FMRP in neural progenitors (Saffary and Xie, 2011) and profilin1, as well as its Drosophila homolog chickadee, but not profilin2 have been implicated in the cellular defects of fragile X syndrome (FXS) pathology (Reeve et al.”

“FMRP represses the RGC to IP transition, maintaining RGCs in an undifferentiated state [52].”

“Specific examples of FMRP control include regulation of the transition from the radial glial cell to the intermediate progenitor [26] and the regulation of olfactory bulb neuron differentiation [27].”

“Saffary et al. found that the loss of FMRP tend to increase the differentiation rate of the radial glial cells (RGCs) into intermediate progenitor cells (IPCs), leading to a significant reduction in RGCs [55].”

“This is quite reasonable because FMRP has been shown to play an important role in maintaining the RGCs, i.e., the main source of neurons during brain development (Saffary and Xie, 2011; Götz and Barde, 2005).”

“During cerebral corticogenesis of the mouse embryo, loss of Fmrp depletes radial glia cells by increasing their differentiation into intermediate progenitor cells [19], and affects the multipolar to bipolar neuronal transition in the cortical plate [20].”

“However, overexpression of PFN1 can correct developmental defects during corticogenesis in these mice [19,32,33].”

“Moreover, PFN1 expression is reduced in FMRP-depleted mice, a classical disease model related to Fragile-X syndrome (FXS), and overexpression of PFN1 can signi fi - cantly rescue the developmental defects in this disease model (18 – 20).”