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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.

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Trans-axonal control of pedunculus and lobe formation. (A–D) Frontal projections of posterior (top) and anterior regions (middle) of MBs marked by Trio (magenta; α’β’, high; and γ, low). Bottom panels show cross-sections of the pedunculus stained for Trio (magenta) and Dlg (green). Bars: (top) 20 µm; (bottom) 2.5 µm. (A’–D’) Top panels show frontal projections of entire MBs marked by FasII (green; αβ, high; and γ, low). Schematics summarize axonal projection phenotypes. Bars, 20 µm. (A) In control nrg14; P[nrg_wt] animals, Trio-positive axons of α’β’ and γ neurons project into anterior lobes. Within the pedunculus, γ, α’β’, and αβ axons are clearly segregated into distinct concentric layers. (A’) αβ axons form medial and vertical lobes. (B) In nrg14; P[nrg180_ΔFIGQY] mutant animals, α’β’ axons fail to project into the pedunculus and form aberrant ball-like projections in the posterior brain. Only γ neurons (also Trio positive; imaged at higher gain settings compared with controls) form anterior lobes. (B’) αβ axons fail to form anterior lobes and form aberrant projections in the posterior brain. (C) Expression of wild-type Nrg180 in α’β’ neurons of nrg14; P[nrg180_ΔFIGQY] mutants restores anterior projections of α’β’ neurons. Minor perturbations of axonal layer organization are evident in the pedunculus. (C’) In these animals, projections of αβ mutant axons are also efficiently rescued and αβ lobes form next to the wild-type Nrg180-expressing α’β’ lobes (asterisk in C). (D) Expression of wild-type Nrg180 in γ neurons of nrg14; P[nrg180_ΔFIGQY] mutants also rescues α’β’ projections. Pedunculus cross-sections reveal aberrant organization of axonal layers, with mutant αβ axons inappropriately in contact with γ axons (arrow). (D’) In these animals, αβ axons grow into the pedunculus to the pedunculus divide (heel, arrow) but fail to form medial or vertical lobes (note the altered appearance of α’β’ lobes in D due to the absence of αβ lobes, indicated by the asterisk). (E) Quantification of α’β’ phenotypes (n = 24, 55, 18, and 30, respectively, in the order of the genotypes indicated). (F) Quantification of αβ phenotypes (n = 44, 61, 69, and 36, respectively, in the order of the genotypes indicated). (G–J and G’–J’) Frontal projections of entire MBs. (G and G’) In nrg14; P[nrg_ΔFERM] mutant animals, axons of α’β’ and αβ neurons form aberrant ball-like projections in the posterior brain and fail to form anterior lobes. (H and H’) Expression of wild-type Nrg180 in α’β’ neurons of nrg14; P[nrg_ΔFERM] mutants does not rescue the MB phenotype. (I and I’) Expression of wild-type Nrg180 in γ neurons of nrg14; P[nrg_ΔFERM] mutants does not rescue the MB phenotype. (J and J’) Expression of wild-type Nrg180 in all MB neurons efficiently rescues axonal projections. Bars, 20 µm. (K) Quantification of the α’β’ phenotypes (n = 44, 61, 28, 31, and 24, respectively, in the order of the genotypes indicated). (L) Quantification of the αβ phenotypes (n = 102, 82, 65, 48, and 30, respectively, in the order of the genotypes indicated).
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fig6: Trans-axonal control of pedunculus and lobe formation. (A–D) Frontal projections of posterior (top) and anterior regions (middle) of MBs marked by Trio (magenta; α’β’, high; and γ, low). Bottom panels show cross-sections of the pedunculus stained for Trio (magenta) and Dlg (green). Bars: (top) 20 µm; (bottom) 2.5 µm. (A’–D’) Top panels show frontal projections of entire MBs marked by FasII (green; αβ, high; and γ, low). Schematics summarize axonal projection phenotypes. Bars, 20 µm. (A) In control nrg14; P[nrg_wt] animals, Trio-positive axons of α’β’ and γ neurons project into anterior lobes. Within the pedunculus, γ, α’β’, and αβ axons are clearly segregated into distinct concentric layers. (A’) αβ axons form medial and vertical lobes. (B) In nrg14; P[nrg180_ΔFIGQY] mutant animals, α’β’ axons fail to project into the pedunculus and form aberrant ball-like projections in the posterior brain. Only γ neurons (also Trio positive; imaged at higher gain settings compared with controls) form anterior lobes. (B’) αβ axons fail to form anterior lobes and form aberrant projections in the posterior brain. (C) Expression of wild-type Nrg180 in α’β’ neurons of nrg14; P[nrg180_ΔFIGQY] mutants restores anterior projections of α’β’ neurons. Minor perturbations of axonal layer organization are evident in the pedunculus. (C’) In these animals, projections of αβ mutant axons are also efficiently rescued and αβ lobes form next to the wild-type Nrg180-expressing α’β’ lobes (asterisk in C). (D) Expression of wild-type Nrg180 in γ neurons of nrg14; P[nrg180_ΔFIGQY] mutants also rescues α’β’ projections. Pedunculus cross-sections reveal aberrant organization of axonal layers, with mutant αβ axons inappropriately in contact with γ axons (arrow). (D’) In these animals, αβ axons grow into the pedunculus to the pedunculus divide (heel, arrow) but fail to form medial or vertical lobes (note the altered appearance of α’β’ lobes in D due to the absence of αβ lobes, indicated by the asterisk). (E) Quantification of α’β’ phenotypes (n = 24, 55, 18, and 30, respectively, in the order of the genotypes indicated). (F) Quantification of αβ phenotypes (n = 44, 61, 69, and 36, respectively, in the order of the genotypes indicated). (G–J and G’–J’) Frontal projections of entire MBs. (G and G’) In nrg14; P[nrg_ΔFERM] mutant animals, axons of α’β’ and αβ neurons form aberrant ball-like projections in the posterior brain and fail to form anterior lobes. (H and H’) Expression of wild-type Nrg180 in α’β’ neurons of nrg14; P[nrg_ΔFERM] mutants does not rescue the MB phenotype. (I and I’) Expression of wild-type Nrg180 in γ neurons of nrg14; P[nrg_ΔFERM] mutants does not rescue the MB phenotype. (J and J’) Expression of wild-type Nrg180 in all MB neurons efficiently rescues axonal projections. Bars, 20 µm. (K) Quantification of the α’β’ phenotypes (n = 44, 61, 28, 31, and 24, respectively, in the order of the genotypes indicated). (L) Quantification of the αβ phenotypes (n = 102, 82, 65, 48, and 30, respectively, in the order of the genotypes indicated).

Mentions: Based on the dynamic expression of Nrg at the border between ingrowing and substrate axons, we hypothesized that Nrg acts as a homophilic CAM to mediate axon–axon interactions during pedunculus entry. To test this hypothesis and to investigate potential cell type–specific requirements of the different Nrg domains, we used the UAS-Gal4 system to express wild-type Nrg180 selectively in either α′β′ or γ neurons in the background of the domain-specific nrg mutants. In these animals, wild-type Nrg180 will be present only in substrate (γ) or ingrowing axons (α′β′) while all other MB neurons express mutant Nrg. This enables a direct analysis of cell type–specific axo–axonal interactions mediated between wild-type and mutant Nrg proteins. In nrg14; P[nrg180_ΔFIGQY], expression of wild-type Nrg180 in ingrowing α′β′ neurons was sufficient to rescue pedunculus entry and lobe formation of α′β′ axons (Fig. 6, A–C and E). Strikingly, in these animals we also observed an almost complete rescue of αβ axons that only express mutant Nrg lacking the FIGQY domain (Fig. 6, A′–C′ and F). Similarly, expression of wild-type Nrg180 in γ neurons was sufficient to rescue projections of mutant α′β′ neurons (Fig. 6, D and E). These data demonstrate that the presence of wild-type Nrg180 in either substrate or ingrowing axons is sufficient to compensate for the absence of the Nrg FIGQY protein interaction motif within the interacting axonal population and indicates that Nrg acts as a homophilic CAM during these axo–axonal interactions. Interestingly, in animals expressing wild-type Nrg180 only in γ neurons, we also observed a partial rescue of αβ projections into the pedunculus, but the axons failed to innervate αβ lobes (Fig. 6, D′ and F). Analysis of the axonal projections within the pedunculus revealed a severe perturbation of axonal layer organization, with mutant αβ neurons now directly contacting wild-type Nrg180-expressing γ neurons (Fig. 6 D, arrow), a phenotype never observed in control animals (Fig. 6 A). Thus, mutant αβ axons likely used wild-type Nrg-expressing γ axons as a substrate to enter the pedunculus. However, at the end of the pedunculus these mutant αβ axons failed to use the nrg14; P[nrg180_ΔFIGQY] mutant α′β′ lobes (Fig. 6, D and D′) as a template and therefore could not form αβ lobes. These data are consistent with the two axonal populations also interacting in an Nrg-dependent manner at this choice point during lobe development.


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)

Trans-axonal control of pedunculus and lobe formation. (A–D) Frontal projections of posterior (top) and anterior regions (middle) of MBs marked by Trio (magenta; α’β’, high; and γ, low). Bottom panels show cross-sections of the pedunculus stained for Trio (magenta) and Dlg (green). Bars: (top) 20 µm; (bottom) 2.5 µm. (A’–D’) Top panels show frontal projections of entire MBs marked by FasII (green; αβ, high; and γ, low). Schematics summarize axonal projection phenotypes. Bars, 20 µm. (A) In control nrg14; P[nrg_wt] animals, Trio-positive axons of α’β’ and γ neurons project into anterior lobes. Within the pedunculus, γ, α’β’, and αβ axons are clearly segregated into distinct concentric layers. (A’) αβ axons form medial and vertical lobes. (B) In nrg14; P[nrg180_ΔFIGQY] mutant animals, α’β’ axons fail to project into the pedunculus and form aberrant ball-like projections in the posterior brain. Only γ neurons (also Trio positive; imaged at higher gain settings compared with controls) form anterior lobes. (B’) αβ axons fail to form anterior lobes and form aberrant projections in the posterior brain. (C) Expression of wild-type Nrg180 in α’β’ neurons of nrg14; P[nrg180_ΔFIGQY] mutants restores anterior projections of α’β’ neurons. Minor perturbations of axonal layer organization are evident in the pedunculus. (C’) In these animals, projections of αβ mutant axons are also efficiently rescued and αβ lobes form next to the wild-type Nrg180-expressing α’β’ lobes (asterisk in C). (D) Expression of wild-type Nrg180 in γ neurons of nrg14; P[nrg180_ΔFIGQY] mutants also rescues α’β’ projections. Pedunculus cross-sections reveal aberrant organization of axonal layers, with mutant αβ axons inappropriately in contact with γ axons (arrow). (D’) In these animals, αβ axons grow into the pedunculus to the pedunculus divide (heel, arrow) but fail to form medial or vertical lobes (note the altered appearance of α’β’ lobes in D due to the absence of αβ lobes, indicated by the asterisk). (E) Quantification of α’β’ phenotypes (n = 24, 55, 18, and 30, respectively, in the order of the genotypes indicated). (F) Quantification of αβ phenotypes (n = 44, 61, 69, and 36, respectively, in the order of the genotypes indicated). (G–J and G’–J’) Frontal projections of entire MBs. (G and G’) In nrg14; P[nrg_ΔFERM] mutant animals, axons of α’β’ and αβ neurons form aberrant ball-like projections in the posterior brain and fail to form anterior lobes. (H and H’) Expression of wild-type Nrg180 in α’β’ neurons of nrg14; P[nrg_ΔFERM] mutants does not rescue the MB phenotype. (I and I’) Expression of wild-type Nrg180 in γ neurons of nrg14; P[nrg_ΔFERM] mutants does not rescue the MB phenotype. (J and J’) Expression of wild-type Nrg180 in all MB neurons efficiently rescues axonal projections. Bars, 20 µm. (K) Quantification of the α’β’ phenotypes (n = 44, 61, 28, 31, and 24, respectively, in the order of the genotypes indicated). (L) Quantification of the αβ phenotypes (n = 102, 82, 65, 48, and 30, respectively, in the order of the genotypes indicated).
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fig6: Trans-axonal control of pedunculus and lobe formation. (A–D) Frontal projections of posterior (top) and anterior regions (middle) of MBs marked by Trio (magenta; α’β’, high; and γ, low). Bottom panels show cross-sections of the pedunculus stained for Trio (magenta) and Dlg (green). Bars: (top) 20 µm; (bottom) 2.5 µm. (A’–D’) Top panels show frontal projections of entire MBs marked by FasII (green; αβ, high; and γ, low). Schematics summarize axonal projection phenotypes. Bars, 20 µm. (A) In control nrg14; P[nrg_wt] animals, Trio-positive axons of α’β’ and γ neurons project into anterior lobes. Within the pedunculus, γ, α’β’, and αβ axons are clearly segregated into distinct concentric layers. (A’) αβ axons form medial and vertical lobes. (B) In nrg14; P[nrg180_ΔFIGQY] mutant animals, α’β’ axons fail to project into the pedunculus and form aberrant ball-like projections in the posterior brain. Only γ neurons (also Trio positive; imaged at higher gain settings compared with controls) form anterior lobes. (B’) αβ axons fail to form anterior lobes and form aberrant projections in the posterior brain. (C) Expression of wild-type Nrg180 in α’β’ neurons of nrg14; P[nrg180_ΔFIGQY] mutants restores anterior projections of α’β’ neurons. Minor perturbations of axonal layer organization are evident in the pedunculus. (C’) In these animals, projections of αβ mutant axons are also efficiently rescued and αβ lobes form next to the wild-type Nrg180-expressing α’β’ lobes (asterisk in C). (D) Expression of wild-type Nrg180 in γ neurons of nrg14; P[nrg180_ΔFIGQY] mutants also rescues α’β’ projections. Pedunculus cross-sections reveal aberrant organization of axonal layers, with mutant αβ axons inappropriately in contact with γ axons (arrow). (D’) In these animals, αβ axons grow into the pedunculus to the pedunculus divide (heel, arrow) but fail to form medial or vertical lobes (note the altered appearance of α’β’ lobes in D due to the absence of αβ lobes, indicated by the asterisk). (E) Quantification of α’β’ phenotypes (n = 24, 55, 18, and 30, respectively, in the order of the genotypes indicated). (F) Quantification of αβ phenotypes (n = 44, 61, 69, and 36, respectively, in the order of the genotypes indicated). (G–J and G’–J’) Frontal projections of entire MBs. (G and G’) In nrg14; P[nrg_ΔFERM] mutant animals, axons of α’β’ and αβ neurons form aberrant ball-like projections in the posterior brain and fail to form anterior lobes. (H and H’) Expression of wild-type Nrg180 in α’β’ neurons of nrg14; P[nrg_ΔFERM] mutants does not rescue the MB phenotype. (I and I’) Expression of wild-type Nrg180 in γ neurons of nrg14; P[nrg_ΔFERM] mutants does not rescue the MB phenotype. (J and J’) Expression of wild-type Nrg180 in all MB neurons efficiently rescues axonal projections. Bars, 20 µm. (K) Quantification of the α’β’ phenotypes (n = 44, 61, 28, 31, and 24, respectively, in the order of the genotypes indicated). (L) Quantification of the αβ phenotypes (n = 102, 82, 65, 48, and 30, respectively, in the order of the genotypes indicated).
Mentions: Based on the dynamic expression of Nrg at the border between ingrowing and substrate axons, we hypothesized that Nrg acts as a homophilic CAM to mediate axon–axon interactions during pedunculus entry. To test this hypothesis and to investigate potential cell type–specific requirements of the different Nrg domains, we used the UAS-Gal4 system to express wild-type Nrg180 selectively in either α′β′ or γ neurons in the background of the domain-specific nrg mutants. In these animals, wild-type Nrg180 will be present only in substrate (γ) or ingrowing axons (α′β′) while all other MB neurons express mutant Nrg. This enables a direct analysis of cell type–specific axo–axonal interactions mediated between wild-type and mutant Nrg proteins. In nrg14; P[nrg180_ΔFIGQY], expression of wild-type Nrg180 in ingrowing α′β′ neurons was sufficient to rescue pedunculus entry and lobe formation of α′β′ axons (Fig. 6, A–C and E). Strikingly, in these animals we also observed an almost complete rescue of αβ axons that only express mutant Nrg lacking the FIGQY domain (Fig. 6, A′–C′ and F). Similarly, expression of wild-type Nrg180 in γ neurons was sufficient to rescue projections of mutant α′β′ neurons (Fig. 6, D and E). These data demonstrate that the presence of wild-type Nrg180 in either substrate or ingrowing axons is sufficient to compensate for the absence of the Nrg FIGQY protein interaction motif within the interacting axonal population and indicates that Nrg acts as a homophilic CAM during these axo–axonal interactions. Interestingly, in animals expressing wild-type Nrg180 only in γ neurons, we also observed a partial rescue of αβ projections into the pedunculus, but the axons failed to innervate αβ lobes (Fig. 6, D′ and F). Analysis of the axonal projections within the pedunculus revealed a severe perturbation of axonal layer organization, with mutant αβ neurons now directly contacting wild-type Nrg180-expressing γ neurons (Fig. 6 D, arrow), a phenotype never observed in control animals (Fig. 6 A). Thus, mutant αβ axons likely used wild-type Nrg-expressing γ axons as a substrate to enter the pedunculus. However, at the end of the pedunculus these mutant αβ axons failed to use the nrg14; P[nrg180_ΔFIGQY] mutant α′β′ lobes (Fig. 6, D and D′) as a template and therefore could not form αβ lobes. These data are consistent with the two axonal populations also interacting in an Nrg-dependent manner at this choice point during lobe development.

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