Cdc42 and Rac1 Signaling Are Both Required for and Act Synergistically in the Correct Formation of Myelin Sheaths in the CNS

“…known for their roles in regulating signaling pathways linking extracellular stimuli to the assembly and organization of the actin cytoskeleton (Hall, 1998), also control microtubule dynamics, cell polarity, membrane trafficking, and gene transcription (Etienne-Manneville and Hall, 2002; Jaffe…”

“To follow the fate of the recombined cells by detection of -galactosidase expression, we bred the conditional LacZ allele from the ROSA26 reporter mouse strain (Soriano, 1999) into control and mutant mice.”

“Cdc42 is not required for OPC proliferation, directed migration, or morphological differentiation Our results do not support a requirement for cdc42 in OPC proliferation, directed migration, or in vitro morphological differentiation; however, there is a significant body of evidence showing a key role for cdc42 in the…”

“Oligodendrocyte progenitor cells (OPCs) were purified from mixed glial cultures (McCarthy and de Vellis, 1980) and differentiated on extracellular matrix substrates (Relvas et al., 2001).”

“…roles in regulating signaling pathways linking extracellular stimuli to the assembly and organization of the actin cytoskeleton (Hall, 1998), also control microtubule dynamics, cell polarity, membrane trafficking, and gene transcription (Etienne-Manneville and Hall, 2002; Jaffe and Hall, 2005).”

“Consequently, in wells to which 25 or 100 ng/ml of netrin-1 was added to the bottom chamber, control OPC migration toward the lower surface of the filter was inhibited significantly ( p 0.01 and 0.009, respectively), as compared with wells to which no netrin-1 was added; however, control and mutant OPCs migrated equal…”

“Cdc42 is not required for OPC proliferation, directed migration, or morphological differentiation Our results do not support a requirement for cdc42 in OPC proliferation, directed migration, or in vitro morphological differentiation; however, there is a significant body of evidence showing a key role for cdc42 in the…”

“This observation raises important questions about the specificity of Rho GTPase dominant-negative approaches and is in line with previous reports showing that dominant-negative mutant expression induced more severe phenotypes than those produced by genetic loss-of-function approaches on identical cell types or organis…”

“…Chrostek,3 Sven M. Krause,1 Klaus-Armin Nave,4 Robin J. M. Franklin, 5 Cord Brakebusch,2 Ueli Suter,1 and João B. Relvas1 1Institute for Cell Biology, Department of Biology, Federal Institute of Technology (ETH) Zurich, CH-8093 Zurich, Switzerland, 2Department of Molecular Pathology, University…”

“Rac1 activity was measured as described previously (Sander et al., 1998).”

“In the context of CNS myelination, pathways known to influence oligodendrocyte development such as those initiated by the activation of integrin receptors (Liang et al., 2004) or the transmembrane protein Lingo-1 (Mi et al., 2005) can modulate the activities of cdc42, rac1, and rhoA.”

“Many key cellular functions ascribed to Rho GTPases rely on experiments during which their function is blocked by expression of dominant-negative mutants (Feig, 1999).”

“During oligodendrocyte development, the coordinated interpretation of extracellular cues, such as integrin, growth factor, or chemokine receptor activation, allows oligodendrocytes to sense their environment and regulate their intrinsic developmental program (Miller, 2002; Baron et al., 2005).”

“Although rhoA is expressed and active during early progenitor stages, the expression and activity of cdc42 and rac1 increase as differentiation proceeds (Liang et al., 2004).”

“, 2004), control and mutant OPCs were allowed to differentiate in culture (Buttery and ffrench-Constant, 1999; Relvas et al., 2001).”

“the CNS Tina Thurnherr,1* Yves Benninger,1* Xunwei Wu,2 Anna Chrostek,3 Sven M. Krause,1 Klaus-Armin Nave,4 Robin J. M. Franklin, 5 Cord Brakebusch,2 Ueli Suter,1 and João B. Relvas1 1Institute for Cell Biology, Department of Biology, Federal Institute of Technology (ETH) Zurich, CH-8093 Zurich, Switzerland, 2Departm…”

“…that the formation of the multilamelar myelin sheath requires the continuous progression of the inner tongue over the axonal surface (Speidel, 1964; Bunge et al., 1989), it is not immediately evident to us how enlarged inner tongues, sometimes more than several axon diameters in length, are still…”

“The generation of conditional Cdc42 mutant mice (Wu et al., 2006) and Rac1 mutant mice (Chrostek et al., 2006) have been described previously.”

“Therefore, dominant-negative mutants of cdc42 may inhibit, especially if expressed at high levels (Braga et al., 2000), the activation of other Rho GTPases, making it nearly impossible to discriminate between the specific contributions from different Rho GTPases.”

“Recent work (Czuchra et al., 2005) has shown that the transfection of Cdc42-null fibroblastoid cells with a Cdc42 dominant-negative mutant (N17 cdc42) elicits additional phenotypes that are not present in untransfected Cdc42null fibroblasts.”

“This conclusion was also supported by standard in vitro migration assays (Boyden chamber; see Material and Methods) with which we tested the capacity of freshly isolated control and mutant OPC to migrate in response to PDGF, an OPC chemoattractant secreted along OPC migratory pathways (McKinnon et al., 1993), or netri…”

“, 1993), or netrin-1, an OPC chemorepellent expressed at the ventricular zone during OPC emigration (Jarjour et al., 2003).”

“The generation of conditional Cdc42 mutant mice (Wu et al., 2006) and Rac1 mutant mice (Chrostek et al., 2006) have been described previously.”

“The sporadic presence of such structures during normal development has been reported previously in amphibian cerebellum (Rosenbluth, 1966).”

“This observation raises important questions about the specificity of Rho GTPase dominant-negative approaches and is in line with previous reports showing that dominant-negative mutant expression induced more severe phenotypes than those produced by genetic loss-of-function approaches on identical cell types or organis…”

“During oligodendrocyte development, the coordinated interpretation of extracellular cues, such as integrin, growth factor, or chemokine receptor activation, allows oligodendrocytes to sense their environment and regulate their intrinsic developmental program (Miller, 2002; Baron et al., 2005).”

“…the myelin abnormalities observed in the Cdc42 mutant CNS could be directly attributed to impairment of oligodendrocyte process extension and branching (Liang et al., 2004), control and mutant OPCs were allowed to differentiate in culture (Buttery and ffrench-Constant, 1999; Relvas et al., 2001).”

“…Krause,1 Klaus-Armin Nave,4 Robin J. M. Franklin, 5 Cord Brakebusch,2 Ueli Suter,1 and João B. Relvas1 1Institute for Cell Biology, Department of Biology, Federal Institute of Technology (ETH) Zurich, CH-8093 Zurich, Switzerland, 2Department of Molecular Pathology, University of Copenhagen, 2100…”

“…it is thought that the formation of the multilamelar myelin sheath requires the continuous progression of the inner tongue over the axonal surface (Speidel, 1964; Bunge et al., 1989), it is not immediately evident to us how enlarged inner tongues, sometimes more than several axon diameters in…”

“In oligodendrocytes, the organization of the microfilament and microtubule array is likely to provide the driving force for changes in plasma membrane conformation associated with process extension and branching (Simpson and Armstrong, 1999; Richter-Landsberg, 2001; Song et al., 2001).”

“These GTPases are expressed by oligodendrocytes (Erschbamer et al., 2005), and, in primary oligodendrocyte cultures, their expression and activity are developmentally regulated (Liang et al., 2004).”

“6C), which also acts as an OPC mitogen (Besnard et al., 1987).”

“Rac1 is required for proper axon ensheathment and myelination Cdc42 and rac1 cooperate in pathways regulating important cellular functions (Cotteret and Chernoff, 2002).”

“The excluded cytoplasm presumably flows to regions of uncompacted myelin, which in the early developing CNS alternate irregularly with compacted myelin sheaths along the individual axon (Storts and Koestner, 1969; Remahl and Hildebrand, 1990).”

“In oligodendrocytes, the organization of the microfilament and microtubule array is likely to provide the driving force for changes in plasma membrane conformation associated with process extension and branching (Simpson and Armstrong, 1999; Richter-Landsberg, 2001; Song et al., 2001).”

“Perturbation of cytoskeletal reorganization and eventually of vesicle transport, also a putative function of cdc42 (Larocca and Rodriguez-Gabin, 2002; RodriguezBoulan et al., 2005), may also explain the presence of large vesicles sometimes found in the cytoplasm of the mutant cells (Figs.”

“The excluded cytoplasm presumably flows to regions of uncompacted myelin, which in the early developing CNS alternate irregularly with compacted myelin sheaths along the individual axon (Storts and Koestner, 1969; Remahl and Hildebrand, 1990).”

“In oligodendrocytes — the glial cells of the central nervous system — myelin sheaths are thinner in the absence of CDC42, which seems to be the result of a defect in removing cytoplasm as the oligodendrocyte plasma membranes wrap around the axo”

“(76) In the case of oligodendrocytes, Cdc42 is important for the correct formation of myelin sheaths.(76) In the case of neuronal precursors, Cdc42 plays crucial roles in the establishment of Par6-dependent apico-basal polarity processes of stem cells.”

“For example, the deletion of Cdc42 or Rac1 in oligodendrocytes results in the formation of myelin outfoldings, but with reduced overall myelin thickness (622).”

“…that govern the cytoskeletal function during developmental myelination in the CNS, namely the β1 integrin, the Rho GTPases Rac1 and Cdc42 and the extracellular matrix (ECM) proteins fibronectin and laminin (Relvas et al., 2001; Benninger et al., 2006; Thurnherr et al., 2006; Lourenco et al., 2016).”