UNC-4/UNC-37-dependent repression of motor neuron-specific genes controls synaptic choice in Caenorhabditis elegans

Abstract

The UNC-4 homeoprotein and the Groucho-like corepressor UNC-37 specify synaptic choice in the Caenorhabditis elegans motor neuron circuit. In unc-4 mutants, VA motor neurons are miswired with inputs from interneurons normally reserved for their lineal sisters, the VB motor neurons. Here we show that UNC-4 and UNC-37 function together in VA motor neurons to repress VB-specific genes and that this activity depends on physical contact between UNC-37 and a conserved Engrailed-like repressor domain (eh1) in UNC-4. Missense mutations in the UNC-4 eh1 domain disrupt interactions between UNC-4 and UNC-37 and result in the loss of UNC-4-dependent repressor activity in vivo. A compensatory amino acid substitution in UNC-37 suppresses specific unc-4 alleles by restoring physical interactions with UNC-4 as well as UNC-4-dependent repression of VB-specific genes. We propose that repression of VB-specific genes by UNC-4 and UNC-37 is necessary for the creation of wild-type inputs to VA motor neurons. The existence of mammalian homologs of UNC-4 and UNC-37 indicates that a similar mechanism could regulate synaptic choice in the vertebrate spinal cord.

Figure 1

unc-4 specifies synaptic input to VA motor neurons. (A) Adjacent VA and VB motor neurons are visualized in this transgenic animal with neuron-specific promoters driving expression of the cyan GFP variant in VA motor neurons (unc-4::CFP) and the yellow GFP mutant protein in VB motor neurons (del-1::YFP) (Miller et al. 1999). (B) Most VA and VB motor neurons arise from a common precursor cell (black circle). In wild-type animals, VA neurons, which extend axons anteriorly, receive inputs from VA-type command interneurons (blue): AVA (chemical synapse and gap junction); AVD and AVE (chemical synapse). VB axons project posteriorly and are innervated by the VB-type interneurons (green): AVB (gap junction); PVC (chemical synapse). (C) In unc-4 mutants, VA neurons are miswired with VB-type inputs (i.e., form gap junctions with AVB). The arrows denote en passant synapses between side-by-side processes in the tightly packed ventral nerve cord and therefore are not indicative of axonal branches.

Figure 2

Comparison of UNC-4-related proteins. (A) Schematic of UNC-4-related proteins showing relative positions of the homeodomains (solid rectangles) and eh1 domains (shaded boxes). A complete sequence of the mouse UNC-4 protein has not been reported. (B) Homeodomain sequences of UNC-4-related proteins. Dots indicate identity to C. elegans UNC-4 sequence. Bold residues show similarity to UNC-4. (C) eh1 domains of nematode UNC-4 proteins (C. elegans and C. briggsae) are most similar to eh1 domains of Engrailed family members. Vertebrate and Drosophila UNC-4 eh1 domains most closely resemble the msh homeoprotein class of eh1 sequences. For nematode UNC-4 eh1 comparison, solid boxes indicate identity to nematode UNC-4 and shaded boxes show similarity. (♦) The position of the invariant phenylalanine; (*) residues mutated in the unc-4 alleles e26, e2307, and e2323. For the vertebrate and Drosophila unc-4 eh1 alignments, the solid boxes indicate identity between the mouse and rat UNC-4 proteins and at least one other protein in the comparison; the shaded boxes show similarity.

Figure 3

unc-4 mutations. (A) Approximate locations of UNC-4 point mutations. (HD) Homeodomain; (eh1) Engrailed-like repressor domain. (B) unc-4 frameshifts and deletions. Vertical bars denote EcoRI sites; boxes represent unc-4 exons; solid boxes show exons encoding the homeodomain (exon five contains the eh1 domain); shaded overlays denote deleted regions. Arrowheads indicate splice donor site mutations in e120 and jd16. Large open rectangles around e2320, e120, and jd16 exons denote frameshifted coding sequence.

Figure 4

del-1::GFP and acr-5::GFP are expressed ectopically in A-type motor neurons of unc-4 and unc-37 mutants. GFP expression (green) was visualized in mid- to late L2 larval animals. Autofluorescent gut granules are yellow. (A–F) The del-1::gfp reporter gene is regulated by 1.8 kb of del-1 upstream sequence. In the ventral nerve cord of wild-type L2 animals, del-1::GFP is expressed in the VB motor neurons but is prematurely expressed in the VAs during this period in unc-37(e262) and in unc-4 mutants. (G–L) The acr-5::gfp reporter gene is regulated by 4.2 kb of acr-5::gfp upstream sequence. acr-5::GFP is expressed in B-type motor neurons (DB, VB) in ventral nerve cords of wild-type animals. In unc-4 and unc-37 mutants, acr-5::GFP is ectopically expressed in A-type motor neurons (DA, VA). Genotypes for each row are indicated at left. unc-37(e262) corresponds to UNC-37(H539Y); unc-4(e2321) corresponds to UNC-4(R197K). (C,I) Enlargements of areas indicated by insets in B and H, respectively. Unlabeled motor neurons in E, F, K, and L are identified at top. Bars, 20 μm. Bar in B applies to B and H; bar in C applies to C–L.

Figure 5

UNC-37(E580K) restores UNC-4-dependent repressor activity to specific unc-4 mutants. (A,C) UNC-37(E580K); UNC-4(G188D). UNC-4 fails to repress the B-type-specific genes in A-type motor neurons in this genetic background. (B,D) UNC-37(E580K); UNC-4(R197K). UNC-37(E580K) suppresses the Unc-4 phenotype by restoring UNC-4 repression of del-1::gfp and acr-5::gfp in A-type motor neurons (cf. Figs. 5, B,D and 4, F,L). Bar, 20 μm.

Figure 6

UNC-4 and UNC-37 interact in yeast two-hybrid assays. UNC-4 proteins are fused to the GAL4 DNA-binding domain; UNC-37 proteins are fused to the GAL4 activation domain. Assays for growth on His⁻ plates were performed in the presence or absence of 3-AT: (+) growth on 100 mm 3-AT; (−) weak or no growth on 100 mm 3-AT. (A) Interactions between full-length UNC-37 and UNC-4 deletion mutants. UNC-4 refers to the full-length protein; UNC-4C represents the carboxy-terminal truncated protein (152–252). Other UNC-4 truncations are denoted by amino acid boundaries. (B) Interactions of UNC-37 and UNC-37(E580K) with UNC-4 eh1 region missense mutations. The allele numbers of previously identified unc-4 mutations are indicated. Asterisks indicate relative positions of mutations. (C) Selected growth assays ± 100 mm 3-AT from A and B.

Figure 7

Model for synaptic choice. (A) In wild-type animals, UNC-4 and UNC-37 prevent the adoption of VB-type presynaptic inputs by repressing VB-specifying genes. (B) In the absence of unc-4 function, VB-specific genes are derepressed in the VAs thereby imposing inputs from VB-type command interneurons.