A multi-protein receptor-ligand complex underlies combinatorial dendrite guidance choices in C. elegans.

Ligand receptor interactions instruct axon guidance during development. How dendrites are guided to specific targets is less understood. The C. elegans PVD sensory neuron innervates muscle-skin interface with its elaborate dendritic branches. Here, we found that LECT-2, the ortholog of leukocyte cell-derived chemotaxin-2 (LECT2), is secreted from the muscles and required for muscle innervation by PVD. Mosaic analyses showed that LECT-2 acted locally to guide the growth of terminal branches. Ectopic expression of LECT-2 from seam cells is sufficient to redirect the PVD dendrites onto seam cells. LECT-2 functions in a multi-protein receptor-ligand complex that also contains two transmembrane ligands on the skin, SAX-7/L1CAM and MNR-1, and the neuronal transmembrane receptor DMA-1. LECT-2 greatly enhances the binding between SAX-7, MNR-1 and DMA-1. The activation of DMA-1 strictly requires all three ligands, which establishes a combinatorial code to precisely target and pattern dendritic arbors

Caenorhabditis elegans Muscleblind homolog mbl-1 functions in neurons to regulate synapse formation

<p>Abstract</p> <p>Background</p> <p>The sequestration of Muscleblind splicing regulators results in myotonic dystrophy. Previous work on Muscleblind has largely focused on its roles in muscle development and maintenance due to the skeletal and cardiac muscle degeneration phenotype observed in individuals with the disorder. However, a number of reported nervous system defects suggest that Muscleblind proteins function in other tissues as well.</p> <p>Results</p> <p>We have identified a mutation in the <it>Caenorhabditis elegans </it>homolog of Muscleblind, <it>mbl-1</it>, that is required for proper formation of neuromuscular junction (NMJ) synapses. <it>mbl-1 </it>mutants exhibit selective loss of the most distal NMJ synapses in a <it>C. elegans </it>motorneuron, DA9, visualized using the vesicle-associated protein RAB-3, as well as the active zone proteins SYD-2/liprin-α and UNC-10/Rim. The proximal NMJs appear to have normal pre- and postsynaptic specializations. Surprisingly, expressing a <it>mbl-1 </it>transgene in the presynaptic neuron is sufficient to rescue the synaptic defect, while muscle expression has no effect. Consistent with this result, <it>mbl-1 </it>is also expressed in neurons.</p> <p>Conclusions</p> <p>Based on these results, we conclude that in addition to its functions in muscle, the Muscleblind splice regulators also function in neurons to regulate synapse formation.</p

MBL-1 and EEL-1 affect the splicing and protein levels of MEC-3 to control dendrite complexity.

Transcription factors (TFs) play critical roles in specifying many aspects of neuronal cell fate including dendritic morphology. How TFs are accurately regulated during neuronal morphogenesis is not fully understood. Here, we show that LIM homeodomain protein MEC-3, the key TF for C. elegans PVD dendrite morphogenesis, is regulated by both alternative splicing and an E3 ubiquitin ligase. The mec-3 gene generates several transcripts by alternative splicing. We find that mbl-1, the orthologue of the muscular dystrophy disease gene muscleblind-like (MBNL), is required for PVD dendrite arbor formation. Our data suggest mbl-1 regulates the alternative splicing of mec-3 to produce its long isoform. Deleting the long isoform of mec-3(deExon2) causes reduction of dendrite complexity. Through a genetic modifier screen, we find that mutation in the E3 ubiquitin ligase EEL-1 suppresses mbl-1 phenotype. eel-1 mutants also suppress mec-3(deExon2) mutant but not the mec-3 null phenotype. Loss of EEL-1 alone leads to excessive dendrite branches. Together, these results indicate that MEC-3 is fine-tuned by alternative splicing and the ubiquitin system to produce the optimal level of dendrite branches

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

Schematic model of the regulation mechanisms of dendrite morphogenesis in <i>C</i>. <i>elegans</i> PVD neuron.

(A) In wild-type animals, MBL-1 regulates the alternative splicing of mec-3, producing the great majority of mec-3a isoform and very few of the mec-3d isoform mRNAs, therefore promotes the normal dendrite matures. (B) In mbl-1 mutant, the ratio of mec-3a isoform and d isoform changes in that the d isoform is predominant, hampering the transcriptional efficiency of mec-3 downstream targets, ultimately decreasing dendrite complexity significantly. (C) In mbl-1 and eel-1 double mutants, the increased MEC-3d isoform quantity due to the E3 ligase EEL-1 mutant, although with lower transcriptional activity, restores the dendritic arbors to a WT-like morphology. Alternatively, EEL-1 functions indirectly to regulate MEC-3.</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

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