Atypical TGF-β signaling controls neuronal guidance in Caenorhabditis elegans

Abstract

Coordinated expression of cell adhesion and signaling molecules is crucial for brain development. Here, we report that the Caenorhabditis elegans transforming growth factor β (TGF-β) type I receptor SMA-6 (small-6) acts independently of its cognate TGF-β type II receptor DAF-4 (dauer formation-defective-4) to control neuronal guidance. SMA-6 directs neuronal development from the hypodermis through interactions with three, orphan, TGF-β ligands. Intracellular signaling downstream of SMA-6 limits expression of NLR-1, an essential Neurexin-like cell adhesion receptor, to enable neuronal guidance. Together, our data identify an atypical TGF-β-mediated regulatory mechanism to ensure correct neuronal development.

Keywords: Biological sciences; Developmental neuroscience; Molecular neuroscience.

Graphical abstract

Figure 1

SMA-6, a TGF-β type I receptor controls HSN development (A) The TGF-β signaling pathways in C. elegans control body size/male tail development (left), and dauer formation (right). Each pathway utilizes a common TGF-β type II receptor (DAF-4), and distinct ligands (DBL-1 and DAF-7), TGF-β type I receptors (SMA-6 and DAF-1), and SMAD transcriptional regulators (SMA-2/3/4 and DAF-3/8/14). TIG-2, TIG-3, and UNC-129 are orphan ligands that have not been assigned to either pathway. Receptors are shown as monomers for simplicity. (B) In wild-type animals (schematic and top panel), HSN cell bodies migrate just posterior to the vulva and extend axons into separate fascicles in the ventral nerve cord. In sma-6(wk7) animals, HSN cell bodies under-migrate, and their axons are misguided (middle and bottom panels). Vulval position is marked with a red asterisk, wild-type positioned cell bodies with white arrowheads, and misguided cell bodies and axons with blue arrowheads. HSNs imaged using the zdIs13(tph-1p::GFP) transgenic strain. Ventral view, anterior to the left. Scale bar: 20 μm. (C) Quantification of HSN developmental defects in TGF-β type I receptor mutants sma-6 and daf-1. Loss of sma-6 but not daf-1 causes HSN developmental defects. Driving sma-6 expression using its own promoter or a hypodermal promoter (elt-3) rescues sma-6(wk7)-induced HSN developmental defects. Driving sma-6 expression using intestinal (ges-1) or HSN (tph-1) promoters does not rescue sma-6(wk7)-induced HSN developmental defects. # refers to independent transgenic lines. n > 100; ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, n.s. not significant (One-way ANOVA with Tukey’s correction). Error bars represent mean ± SEM. (D) Quantification of HSN developmental defects in animals lacking SMAD transcriptional regulators. Loss of SMADs that control body size and male tail development (SMA-2/3/4) but not dauer SMADs (DAF-3/8/14) causes HSN developmental defects. n > 100; ∗∗p < 0.01, ∗∗∗∗p < 0.0001, n.s. not significant (One-way ANOVA with Tukey’s correction). Error bars represent mean ± SEM.

Figure 2

DAF-4, the sole C. elegans TGF-β type II receptor, is dispensable for HSN development (A) Protein domain organization of the DAF-4 TGF-β type II receptor and mutant alleles used in this study. All genetic lesions (red lines) cause frameshifts. SP = signal peptide; TM = transmembrane domain. (B) Quantification of HSN developmental defects in wild-type and daf-4 mutant animals. All daf-4 mutant alleles exhibit wild-type HSN development. n > 100; n.s. not significant (One-way ANOVA with Tukey’s correction). Error bars represent mean ± SEM. (C) Quantification of HSN developmental defects in wild-type, sma-6(wk7), rme-1(b1045), arf-6(tm1447), and rme-1; sma-6 mutant animals. Loss of retromer-dependent SMA-6 recycling but not ARF-6-dependent DAF-4 recycling causes HSN developmental defects. n > 100; ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, n.s. not significant (One-way ANOVA with Tukey’s correction). Error bars represent mean ± SEM. (D) Quantification of HSN developmental defects in wild-type, sma-6(wk7), daf-3(mgDf90), and sma-6; daf-3 mutant animals. n > 100; n.s. not significant (One-way ANOVA with Tukey’s correction). Error bars represent mean ± SEM. (E) Quantification of HSN developmental defects in wild-type, sma-6(wk7), daf-14(m77), and sma-6; daf-14 mutant animals. n > 100; n.s. not significant (One-way ANOVA with Tukey’s correction). Error bars represent mean ± SEM Note: the mgIs71(tph-1p::GFP) transgene was used for HSN analysis in (E) which has a lower background HSN phenotype compared to zdIs13(tph-1p::GFP).

Figure 3

SMA-6 interacts with three, orphan, TGF-β ligands to control HSN development (A) Quantification of HSN developmental defects in dbl-1(wk70), daf-7(e1372), tig-2(ok3416), tig-3(tm2092), and unc-129(ev554) single mutants, and all compound mutant combinations of tig-2, tig-3, and unc-129. dbl-1 and daf-7 mutant animals exhibit wild-type HSN development. Loss of either tig-2, tig-3, or unc-129 causes HSN developmental defects and the tig-2; tig-3; unc-129 triple mutant and each double mutant combination is not significantly different from each single mutant. n > 100; ∗p < 0.05, ∗∗p < 0.01, n.s. not significant (One-way ANOVA with Tukey’s correction). Error bars represent mean ± SEM. (B) Quantification of HSN developmental defects in sma-6(wk7) animals in combination with either tig-2(ok3416), tig-3(tm2092), or unc-129(ev554) mutations. Each double mutant combination is not significantly different from the respective single mutant. n > 100; ∗∗∗∗p < 0.0001, n.s. not significant (One-way ANOVA with Tukey’s correction). Error bars represent mean ± SEM. (C–E) Input (total cell lysates) and immunoprecipitates (IP) from transfected HEK293T cells. Proteins detected with antibodies in western blots (WB) as indicated. (C) TIG-2-FLAG co-precipitates with SMA-6-MYC; (D) TIG-3-HA co-precipitates with SMA-6-MYC; (E) UNC-129-V5 co-precipitates with SMA-6-MYC. kD, kilodalton. Immunoprecipitated SMA-6-MYC is marked with a red arrow. Whole blots in Figure S4.

Figure 4

SMA-3 regulation of the NLR-1 Neurexin-like receptor controls HSN development (A) Heatmap of whole-animal transcriptional profiling from sma-3(wk30) L2 larvae compared to wild-type. Gene names of candidate HSN regulators on the left. Each column represents individual total RNA samples. Downregulated (blue), upregulated (red),and unchanged (white). (B) Quantification of HSN developmental defects in wild-type animals following RNAi knockdown of genes downregulated in the sma-3(wk30) mutant. n > 100; n.s. not significant (One-way ANOVA with Tukey’s correction). Error bars represent mean ± SEM. (C) Quantification of HSN developmental defects in sma-3(wk30) animals following RNAi knockdown of genes upregulated in the sma-3(wk30) mutant. n > 100; ∗∗p < 0.01, n.s. not significant (One-way ANOVA with Tukey’s correction). Error bars represent mean ± SEM. (D) Relative nlr-1 mRNA levels in sma-3(wk30) (left) and sma-6(wk7) (right) mutant L2 larvae normalized to values for wild-type worms. Three biological replicates were compared (cdc-42 reference gene was used). ∗p < 0.05 (t test). Error bars represent mean ± SEM. (E) Quantification of HSN developmental defects in animals overexpressing nlr-1 in the hypodermis (elt-3 promoter) or intestine (ges-1 promoter). nlr-1 overexpression in the hypodermis but not the intestine causes HSN defects. HSN defects of sma-3(wk30) animals are not enhanced by nlr-1 overexpression in the hypodermis. n > 100; ∗∗∗p < 0.001, n.s. not significant (One-way ANOVA with Tukey’s correction). Error bars represent mean ± SEM # refers to independent transgenic lines (line #1 in the sma-3(wk30) background is the same line used in wild-type). (F) HSN developmental defects caused by overexpression of nlr-1 in the hypodermis are phenotypically similar to those observed with loss of sma-6 and sma-3 (see Figure 1B and Table S1). Vulval position is marked with a red asterisk; wild-type positioned cell bodies with white arrowheads, and misguided cell bodies and axons with blue arrowheads. Ventral view, anterior to the left. Scale bar: 20 μm. (G) The TGF-β ligands TIG-2 (red), TIG-3 (green), and UNC-129 (yellow) regulate the TGF-β type I receptor SMA-6 to control HSN guidance non-cell-autonomously from the hypodermis. We hypothesize that TIG-2 homodimers expressed from neurons and TIG-3/UNC-129 homo- or heterodimers expressed from muscle interact with SMA-6. In the hypodermis TGF-β signaling through the SMA-2/3/4 transcriptional regulators limits expression of the Neurexin-like cell adhesion molecule NLR-1 to enable faithful HSN development.