Limits...
L1CAM/Neuroglian controls the axon-axon interactions establishing layered and lobular mushroom body architecture.

Siegenthaler D, Enneking EM, Moreno E, Pielage J - J. Cell Biol. (2015)

Bottom Line: We demonstrate that the Drosophila melanogaster L1CAM homologue Neuroglian mediates adhesion between functionally distinct mushroom body axon populations to enforce and control appropriate projections into distinct axonal layers and lobes essential for olfactory learning and memory.For functional cluster formation, intracellular Ankyrin2 association is sufficient on one side of the trans-axonal complex whereas Moesin association is likely required simultaneously in both interacting axonal populations.Together, our results provide novel mechanistic insights into cell adhesion molecule-mediated axon-axon interactions that enable precise assembly of complex neuronal circuits.

View Article: PubMed Central - HTML - PubMed

Affiliation: Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland University of Basel, 4003 Basel, Switzerland.

Show MeSH

Related in: MedlinePlus

Nrg–Ank2 association controls MB axon guidance. (A–C and F–H) Frontal projections of the anterior (A, B, F, and G), posterior (C), or entire (H) region of the MBs visualized using FasII (green, αβ axons) and Dlg (magenta, neuropil). Bars, 20 µm. (A) In hemizygous nrg305 mutant animals, αβ axons display branching and lobe formation defects but rarely fail to project through the pedunculus. (B) Heterozygous mutations of ank2 (ank2518/+) do not affect MB development. (C) Removal of one copy of ank2 in hemizygous nrg305 mutant animals severely enhances the MB axon phenotype, with MB axons failing to enter the pedunculus and forming aberrant ball-like structures in the posterior brain. (D) Quantification of the αβ axon phenotype assayed using FasII (n = 60, 43, 42, and 44, respectively, in the order of the genotypes given). (E) Western blot analysis of Nrg expression in larval brain extracts. The nrg305 GFP-trap mutation reduces protein expression of both Nrg180 and Nrg167. (F) nrg305 mutant animals display mild axonal defects including branching and lobe formation defects. (G) Knockdown of Moesin in MB neurons causes defects in αβ axon branching and lobe formation but does not lead to aberrant axonal accumulations in the posterior brain. (H) Knockdown of Moesin in MB neurons in nrg305 mutant animals results in a dramatic enhancement of the phenotype compared with both individual genotypes, with MB axons now forming aberrant ball-like structure in the posterior brain. (I) Quantification of αβ axon phenotype using FasII (n = 43, 38, 30, 72, 26, 26, and 26, respectively, in the order of the genotypes indicated). (J) Schematic model indicating essential Nrg interaction partners.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4384726&req=5

fig5: Nrg–Ank2 association controls MB axon guidance. (A–C and F–H) Frontal projections of the anterior (A, B, F, and G), posterior (C), or entire (H) region of the MBs visualized using FasII (green, αβ axons) and Dlg (magenta, neuropil). Bars, 20 µm. (A) In hemizygous nrg305 mutant animals, αβ axons display branching and lobe formation defects but rarely fail to project through the pedunculus. (B) Heterozygous mutations of ank2 (ank2518/+) do not affect MB development. (C) Removal of one copy of ank2 in hemizygous nrg305 mutant animals severely enhances the MB axon phenotype, with MB axons failing to enter the pedunculus and forming aberrant ball-like structures in the posterior brain. (D) Quantification of the αβ axon phenotype assayed using FasII (n = 60, 43, 42, and 44, respectively, in the order of the genotypes given). (E) Western blot analysis of Nrg expression in larval brain extracts. The nrg305 GFP-trap mutation reduces protein expression of both Nrg180 and Nrg167. (F) nrg305 mutant animals display mild axonal defects including branching and lobe formation defects. (G) Knockdown of Moesin in MB neurons causes defects in αβ axon branching and lobe formation but does not lead to aberrant axonal accumulations in the posterior brain. (H) Knockdown of Moesin in MB neurons in nrg305 mutant animals results in a dramatic enhancement of the phenotype compared with both individual genotypes, with MB axons now forming aberrant ball-like structure in the posterior brain. (I) Quantification of αβ axon phenotype using FasII (n = 43, 38, 30, 72, 26, 26, and 26, respectively, in the order of the genotypes indicated). (J) Schematic model indicating essential Nrg interaction partners.

Mentions: Phosphorylation of the tyrosine residue within the FIGQY motif negatively regulates the binding of L1 protein family members to Ankyrins (Garver et al., 1997; Tuvia et al., 1997). This effect can be mimicked by specific amino acid substitutions, which alter binding to Ank2 and change the mobility of L1CAM in vitro (Gil et al., 2003) or of Nrg in axons in vivo (Enneking et al., 2013). Similar to nrg14; P[nrg180_ΔFIGQY] mutant animals, a point mutation abolishing the Nrg–Ank2 interaction (YA) failed to rescue MB development (nrg14; P[nrg180_YA]; Fig. 4 G). In contrast, a point mutation inducing a constitutive Nrg–Ank2 interaction by rendering the tyrosine nonphosphorylatable (YF) efficiently restored MB axon projections into the pedunculus and the anterior lobes (nrg14; P[nrg180_YF]; Fig. 4, F and G). Interestingly, in YF mutants we observed minor defects in α lobe tip innervation and a partial fusion of the β lobes from the two brain hemispheres, which indicates that a dynamic regulation of the Nrg–Ank2 interaction is essential for normal lobe morphogenesis (Fig. 4 F; and Fig. S2, A–C). To independently test the requirement of a cytoplasmic Nrg–Ank2 association, we performed genetic interactions assays. The nrg allele nrg305 significantly reduced expression of both Nrg isoforms (Fig. 5 E) and caused MB lobe formation defects in 65% of brain hemispheres but only mildly affected axonal projection into the pedunculus (Fig. 5, A and D). Strikingly, removal of one copy of ank2 (using the ank2- allele ank2518) in hemizygous nrg305 mutant animals resulted in a dramatic enhancement of the phenotype, with 80% of αβ axons now failing to enter the pedunculus (Fig. 5, C and D). Because the ank2518 allele did not impair MB development in heterozygosity (Fig. 5, B and D), these data are consistent with the Nrg–Ank2 interaction contributing to MB axon guidance (Fig. 5 J).


L1CAM/Neuroglian controls the axon-axon interactions establishing layered and lobular mushroom body architecture.

Siegenthaler D, Enneking EM, Moreno E, Pielage J - J. Cell Biol. (2015)

Nrg–Ank2 association controls MB axon guidance. (A–C and F–H) Frontal projections of the anterior (A, B, F, and G), posterior (C), or entire (H) region of the MBs visualized using FasII (green, αβ axons) and Dlg (magenta, neuropil). Bars, 20 µm. (A) In hemizygous nrg305 mutant animals, αβ axons display branching and lobe formation defects but rarely fail to project through the pedunculus. (B) Heterozygous mutations of ank2 (ank2518/+) do not affect MB development. (C) Removal of one copy of ank2 in hemizygous nrg305 mutant animals severely enhances the MB axon phenotype, with MB axons failing to enter the pedunculus and forming aberrant ball-like structures in the posterior brain. (D) Quantification of the αβ axon phenotype assayed using FasII (n = 60, 43, 42, and 44, respectively, in the order of the genotypes given). (E) Western blot analysis of Nrg expression in larval brain extracts. The nrg305 GFP-trap mutation reduces protein expression of both Nrg180 and Nrg167. (F) nrg305 mutant animals display mild axonal defects including branching and lobe formation defects. (G) Knockdown of Moesin in MB neurons causes defects in αβ axon branching and lobe formation but does not lead to aberrant axonal accumulations in the posterior brain. (H) Knockdown of Moesin in MB neurons in nrg305 mutant animals results in a dramatic enhancement of the phenotype compared with both individual genotypes, with MB axons now forming aberrant ball-like structure in the posterior brain. (I) Quantification of αβ axon phenotype using FasII (n = 43, 38, 30, 72, 26, 26, and 26, respectively, in the order of the genotypes indicated). (J) Schematic model indicating essential Nrg interaction partners.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4384726&req=5

fig5: Nrg–Ank2 association controls MB axon guidance. (A–C and F–H) Frontal projections of the anterior (A, B, F, and G), posterior (C), or entire (H) region of the MBs visualized using FasII (green, αβ axons) and Dlg (magenta, neuropil). Bars, 20 µm. (A) In hemizygous nrg305 mutant animals, αβ axons display branching and lobe formation defects but rarely fail to project through the pedunculus. (B) Heterozygous mutations of ank2 (ank2518/+) do not affect MB development. (C) Removal of one copy of ank2 in hemizygous nrg305 mutant animals severely enhances the MB axon phenotype, with MB axons failing to enter the pedunculus and forming aberrant ball-like structures in the posterior brain. (D) Quantification of the αβ axon phenotype assayed using FasII (n = 60, 43, 42, and 44, respectively, in the order of the genotypes given). (E) Western blot analysis of Nrg expression in larval brain extracts. The nrg305 GFP-trap mutation reduces protein expression of both Nrg180 and Nrg167. (F) nrg305 mutant animals display mild axonal defects including branching and lobe formation defects. (G) Knockdown of Moesin in MB neurons causes defects in αβ axon branching and lobe formation but does not lead to aberrant axonal accumulations in the posterior brain. (H) Knockdown of Moesin in MB neurons in nrg305 mutant animals results in a dramatic enhancement of the phenotype compared with both individual genotypes, with MB axons now forming aberrant ball-like structure in the posterior brain. (I) Quantification of αβ axon phenotype using FasII (n = 43, 38, 30, 72, 26, 26, and 26, respectively, in the order of the genotypes indicated). (J) Schematic model indicating essential Nrg interaction partners.
Mentions: Phosphorylation of the tyrosine residue within the FIGQY motif negatively regulates the binding of L1 protein family members to Ankyrins (Garver et al., 1997; Tuvia et al., 1997). This effect can be mimicked by specific amino acid substitutions, which alter binding to Ank2 and change the mobility of L1CAM in vitro (Gil et al., 2003) or of Nrg in axons in vivo (Enneking et al., 2013). Similar to nrg14; P[nrg180_ΔFIGQY] mutant animals, a point mutation abolishing the Nrg–Ank2 interaction (YA) failed to rescue MB development (nrg14; P[nrg180_YA]; Fig. 4 G). In contrast, a point mutation inducing a constitutive Nrg–Ank2 interaction by rendering the tyrosine nonphosphorylatable (YF) efficiently restored MB axon projections into the pedunculus and the anterior lobes (nrg14; P[nrg180_YF]; Fig. 4, F and G). Interestingly, in YF mutants we observed minor defects in α lobe tip innervation and a partial fusion of the β lobes from the two brain hemispheres, which indicates that a dynamic regulation of the Nrg–Ank2 interaction is essential for normal lobe morphogenesis (Fig. 4 F; and Fig. S2, A–C). To independently test the requirement of a cytoplasmic Nrg–Ank2 association, we performed genetic interactions assays. The nrg allele nrg305 significantly reduced expression of both Nrg isoforms (Fig. 5 E) and caused MB lobe formation defects in 65% of brain hemispheres but only mildly affected axonal projection into the pedunculus (Fig. 5, A and D). Strikingly, removal of one copy of ank2 (using the ank2- allele ank2518) in hemizygous nrg305 mutant animals resulted in a dramatic enhancement of the phenotype, with 80% of αβ axons now failing to enter the pedunculus (Fig. 5, C and D). Because the ank2518 allele did not impair MB development in heterozygosity (Fig. 5, B and D), these data are consistent with the Nrg–Ank2 interaction contributing to MB axon guidance (Fig. 5 J).

Bottom Line: We demonstrate that the Drosophila melanogaster L1CAM homologue Neuroglian mediates adhesion between functionally distinct mushroom body axon populations to enforce and control appropriate projections into distinct axonal layers and lobes essential for olfactory learning and memory.For functional cluster formation, intracellular Ankyrin2 association is sufficient on one side of the trans-axonal complex whereas Moesin association is likely required simultaneously in both interacting axonal populations.Together, our results provide novel mechanistic insights into cell adhesion molecule-mediated axon-axon interactions that enable precise assembly of complex neuronal circuits.

View Article: PubMed Central - HTML - PubMed

Affiliation: Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland University of Basel, 4003 Basel, Switzerland.

Show MeSH
Related in: MedlinePlus