The Bergmann Glia Fibers Orchestrate the Precise Innervation of Stellate Axons to the Purkinje Dendrites

<p>Purkinje neurons (yellow) receive GABAergic inputs from stellate interneurons (blue) exclusively at the dendrites. This precision at the level of partner selection and subcellular localization of synapses is critical for the proper functioning of these cerebellar GABAergic circuits. How is this precision directed during development? In this issue of <i>PLoS Biology</i>, Ango et al. report that Bergmann glia (red) are the central orchestrators in the assembly of this circuit. Bergmann glia act as guideposts, directing the stellate interneuron process to their Purkinje neuron targets and coordinating the development of this precisely wired circuit.</p

Sequence alignment of zinc finger domains between the <i>C</i>. <i>elegans</i> MBL-1, <i>Drosophila</i> muscleblind, and human muscleblind-like protein families.

The two C3H-type zinc finger domains are marked by underlines. A single point mutation in mbl-1(wy888) leads to histidine to tyrosine (H131Y) substitution. (EPS)</p

The HECT ubiquitin ligase domain might be essential for <i>eel-1</i>’s function in suppressing <i>mec-3(deExon2)</i> phenotype.

(A) Schematic of the C. elegans EEL-1 protein sequences. Conserved protein domains are annotated as follows: DUF, domain of unknown function; UBA, ubiquitin-associated domain; CAD, conserved acidic domain; HECT, homologous to E6AP c-terminus domain. The HECT domain is the catalytic ubiquitin ligase domain of EEL-1 protein. In eel-1(wy50891) mutants, the HECT domain is destructed by a frameshift mutation. (B) Representative confocal images showing the PVD dendrite pattern in mec-3(deExon2) and mec-3(deExon2); eel-1(wy50891). Scale bars, 50 μm. (C) Quantification of 2°, 3°, and 4° dendrite number in mec-3(deExon2) and mec-3(deExon2); eel-1(wy50891). Data are shown as mean ± SEM. ***p t test. n>30 for each genotype. (EPS)</p

Expression level changes of the downstream target genes of MEC-3 in <i>mbl-1(wy888)</i> and <i>mbl-1(wy888); eel-1(wy50554)</i>.

(A) qPCR results of acp-2 in WT, mbl-1(wy888), and mbl-1(wy888); eel-1(wy50554). (B) qPCR results of hpo-30 in WT, mbl-1(wy888), and mbl-1(wy888); eel-1(wy50554). (C) qPCR results of T24F1.4 in WT, mbl-1(wy888), and mbl-1(wy888); eel-1(wy50554). (D) qPCR results of egl-46 in WT, mbl-1(wy888), and mbl-1(wy888); eel-1(wy50554). Data are shown as mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test. Not significant (NS) p>0.05, *p (EPS)</p

Diagram of the mutation sites in different <i>eel-1</i> alleles.

Mutation sites of eel-1(wy50554), eel-1(wy50784), eel-1(wy50785), eel-1(wy50786), eel-1(wy50884), eel-1(wy50885), eel-1(wy50886), and eel-1(wy50891) are shown in the diagram. (EPS)</p

<i>egl-46</i> might function in the downstream of <i>mbl-1</i>.

(A) Images showing the PVD dendrite pattern in mbl-1(wy888), egl-46(gk692), and mbl-1(wy888); egl-46(gk692). Scale bars, 50 μm. (B) Quantification of the number of 2°, 3°, and 4° dendrites in mbl-1(wy888) and mbl-1(wy888); PVD::egl-46 animals. Data are shown as mean ± SEM. *pt test. n>20 for each genotype. (EPS)</p

The second exon of MEC-3 locates at the highly conserved N-terminal portion of the LIM1 domain.

The second exon of mec-3 corresponds to AA27-43 of the MEC-3a protein. It locates at the N-terminal portion of the LIM1 domain, which is highly conserved. The exon 2 skipping will cause an in-frame deletion, which disrupts the first LIM domain. (EPS)</p

EEL-1 maintains the normal PVD dendrite through MEC-3.

(A) Representative confocal images showing the PVD dendrite pattern in mec-3(deExon2), mec-3(deExon2); eel-1(wy50785), mec-3(deExon2); PVD::mec-3(deExon2) cDNA, mec-3(wy50748), mec-3(wy50748); PVD::mec-3(deExon2), mec-3(wy50748); eel-1(wy50786). Scale bars, 50 μm. (B-D) Quantification of 2°, 3°, and 4° dendrite number in mec-3(deExon2), mec-3(deExon2); eel-1(wy50785), mec-3(deExon2); PVD::mec-3(deExon2), mec-3(wy50748), mec-3(wy50748); PVD::mec-3(deExon2) cDNA, and mec-3(wy50748); eel-1(wy50786). Data are shown as mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test. Not significant (NS) p>0.05, ***p20 for each genotype.</p

Genetic analysis of <i>mbl-1</i> and genes regulating PVD dendrite.

(A) Double mutant analysis between mbl-1(wy888) and mutants that showed PVD dendrite phenotype. Images showing the PVD dendrite pattern in mbl-1(wy888), dma-1(wy686), mbl-1(wy888); dma-1(wy686), hpo-30(ok2047), mbl-1(wy888); hpo-30(ok2047), kpc-1(gk8), mbl-1(wy888); kpc-1(gk8), tiam-1(tm1556), mbl-1(wy888); tiam-1(tm1556), ire-1(ok799), mbl-1(wy888); ire-1(ok799). Scale bars, 50 μm. (B) A schematic diagram showing the ire-1 genomic locus. The size of RT-PCR products: primer (L1+R1), 1234 bp; primer (L2+R2), 795 bp; primer (L3+R3), 1009 bp. (C) A representative gel of the ire-1 RT-PCR products amplified from the cDNA of WT and mbl-1(wy888) animals. (EPS)</p

E3 ligase EEL-1 mutant suppresses <i>mbl-1(wy888)</i> phenotype.

(A) Representative confocal images showing the PVD dendrite pattern in WT, mbl-1(wy888), eel-1(wy50554), eel-1(wy50554); mbl-1(wy888), eel-1(wy50784); mbl-1(wy888). Scale bars, 50 μm. (B-D) Quantification of 2°, 3°, and 4° dendrite number in WT, mbl-1(wy888), eel-1(wy50554), eel-1(wy50554); mbl-1(wy888), and eel-1(wy50784); mbl-1(wy888). Data are shown as mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test. Not significant (NS) p>0.05, ***p20 for each genotype. (E) A representative gel of the RT-PCR products amplified from the cDNA of WT and eel-1(wy50554); mbl-1(wy888) animals.</p