Integrins Have Cell-Type-Specific Roles in the Development of Motor Neuron Connectivity

Abstract

Formation of the nervous system requires a complex series of events including proper extension and guidance of neuronal axons and dendrites. Here we investigate the requirement for integrins, a class of transmembrane cell adhesion receptors, in regulating these processes across classes of C. elegans motor neurons. We show α integrin/ina-1 is expressed by both GABAergic and cholinergic motor neurons. Despite this, our analysis of hypomorphic ina-1(gm144) mutants indicates preferential involvement of α integrin/ina-1 in GABAergic commissural development, without obvious involvement in cholinergic commissural development. The defects in GABAergic commissures of ina-1(gm144) mutants included both premature termination and guidance errors and were reversed by expression of wild type ina-1 under control of the native ina-1 promoter. Our results also show that α integrin/ina-1 is important for proper outgrowth and guidance of commissures from both embryonic and post-embryonic born GABAergic motor neurons, indicating an ongoing requirement for integrin through two phases of GABAergic neuron development. Our findings provide insights into neuron-specific roles for integrin that would not be predicted based solely upon expression analysis.

Keywords: C. elegans; GABAergic motor neuron; axon guidance; axon outgrowth; commissure; ina-1; integrin.

Figure 1

Commissural defects of ina-1(gm144) mutants. (A) Schematic (far left) and representative confocal image of wild type L4 GABAergic (GABA, red) and cholinergic (ACh, green) motor neuron somas, commissures, ventral nerve cord (VNC), dorsal nerve cord (DNC), and lateral nerve cord (LNC). Right: confocal images of GABAergic and cholinergic commissures in ina-1(gm144) L4 animals. Some GABAergic commissures prematurely bifurcate (arrow) and others prematurely turn prior to reaching the dorsal nerve cord (arrowhead). Scale bar, 10 µm. (B) Diagram of wild type commissural patterning and commissural defects scored. Wild type commissures fully extend between the ventral nerve cord and the dorsal nerve cord. In some ina-1(gm144) animals, commissures fail to reach the dorsal nerve cord, bifurcating prematurely (bottom left panel), turning inappropriately (bottom middle panel) or terminating prematurely (bottom right panel). These phenotypes were collectively scored as incomplete commissures. (C) Quantification of the percentage of incomplete commissures in wild type and ina-1(gm144) animals. The percentage of incomplete GABAergic, but not cholinergic, commissures in ina-1(gm144) animals is significantly increased compared to wild type. Fisher’s exact test, ** p < 0.0002. (D) The percentage of animals with incomplete GABAergic commissures (1 or more commissure defects) is significantly higher in ina-1(gm144) animals compared with wild type. Fisher’s exact test, ** p < 0.0002. Error bars are standard error of the proportion.

Figure 2

Expression of wild type α integrin/ina-1 in ina-1(gm144) mutants rescues GABAergic commissural outgrowth and guidance. Commissural defects were assessed in L4 stage ina-1(gm144) mutants expressing wild type ina-1. Native refers to expression of ina-1 cDNA using ~4.0 kb of regulatory sequence upstream of the ina-1 start. * p < 0.03, ** p < 0.001, Fisher’s exact test with correction for multiple comparisons. Error bars are standard error of the proportion.

Figure 3

ina-1 is expressed in cholinergic and GABAergic motor neurons. (A–C) Confocal images of the ventral nerve cord of animals expressing a transcriptional ina-1 reporter (Pina-1::GFP, green) and GABAergic neuron marker (Punc-47::mCherry, red). (A) Pina-1::GFP is weakly expressed in DD GABAergic motor neurons during the L1 stage. Scale bar, 10 µm. Inset, magnified image of ina-1 expression in DD GABAergic neuron. Scale bar, 5 µm. (B) High levels of epidermal expression during the L1 stage complicate visualization of neuronal fluorescence. Scale bar, 5 µm. (C) Pina-1::GFP is weakly expressed in GABAergic somas at the L4 stage (dashed box). Pina-1::GFP expression is also visible in other ventral nerve cord motor neurons (indicated by asterisks). Scale bar, 10 µm. Inset, high magnification image of Pina-1::GFP expression in GABAergic VD5 motor neuron soma (red outline). Intensity was increased to aid in visualization. Scale bar, 5 µm (D–E). Confocal images of the ventral nerve cord in animals expressing Pina-1::GFP together with the Pacr-2::mCherry cholinergic motor neuron marker (ACh, red). (D) Pina-1::GFP is expressed in a subset of cholinergic motor neurons at the L1 stage (dashed box). Scale bar, 10 µm. Inset, magnified image of Pina-1::GFP expression in L1 stage cholinergic motor neuron somas (red outline). Scale bar, 5 µm. (E) Pina-1::GFP is strongly expressed in a subset of cholinergic motor neurons (likely B-type cholinergic neurons) at the L4 stage. Asterisks denote Pina-1::GFP expression in additional ventral nerve cord neurons not labeled by Pacr-2::mCherry. Some of these may be AS neurons that are not efficiently labeled by this marker. Scale bar, 10 µm. Inset, magnified image of Pina-1::GFP expression in L4 stage cholinergic motor neuron somas (red outline). Scale bar, 5 µm.

Figure 4

INA-1 localizes to the somas and neurites of GABAergic and cholinergic neurons. (A–E) Confocal images of transgenic strains expressing INA-1::GFP with the GABAergic motor neuron marker Punc-47::mCherry (GABA, red). (A) In newly hatched L1s, INA-1::GFP is weakly localized to the cell bodies of DD neurons (dashed box, magnified in B). Asterisks indicate GABAergic neurons expressing INA-1::GFP. Dashed outlines indicate the pharynx. Solid rectangle indicates commissure, magnified in C. Note that INA-1::GFP is also visible in additional motor neuron classes. Scale bar, 10 µm. (B) Magnified view of INA-1::GFP expression in L1 stage ventral cord motor neurons. GABAergic motor neuron outlined in red. Scale bar, 10 µm. (C) Magnified view of GABAergic commissure (dashed white box in A). INA-1::GFP localization is detectable in some GABAergic commissures (white arrows). Scale bar, 5 µm. During L4, INA-1::GFP localizes to GABAergic somas (D,F) and neurites (E,G) (single confocal section) within the ventral nerve cord. Scale bars, 5 µm. (F,G) Quantification of fluorescence intensity in GABAergic and cholinergic ventral nerve cord motor neuron somas (F) and processes (G). (F) INA-1::GFP signal is significantly higher in cholinergic somas compared to GABAergic. GABAergic somas (n = 17), cholinergic somas (n = 22), unpaired t-test * p ≤ 0.05. (G) INA-1::GFP signal is significantly higher in GABAergic processes compared to cholinergic. GABAergic (n = 14), ACh (n = 13), unpaired t-test, ** p ≤ 0.01. (H–J) Confocal images of transgenic strains expressing INA-1::GFP together with the cholinergic motor neuron marker Pacr-2::mCherry. (H) In L1, INA-1::GFP is present in a subset of cholinergic somas (asterisks). Dashed box region is magnified in inset. Scale bar, 20 µm. Inset, magnified view of L1 ventral nerve cord showing INA-1::GFP localization in a subset of cholinergic neurons (red outlines). Scale bar, 5 µm. (I) INA-1::GFP is expressed at varying levels in most cholinergic somas at the L4 stage. Scale bar, 5 µm. (J) Single confocal section of ventral nerve cord in L4 animals. INA-1::GFP is diffusely localized in cholinergic neurites but stronger INA-1::GFP fluorescence is evident in an adjacent subset of neurites not labeled by the Pacr-2::mCherry cholinergic motor neuron reporter. Based on 4G, these are likely to be GABAergic neurites. Scale bar, 5 µm. Bottom, cartoon representation of confocal images (VNC = ventral nerve cord).

Figure 5

ina-1 is important for commissural patterning of both embryonic and post-embryonic born GABAergic neurons. (A–C) GABAergic commissures were visualized in animals expressing Punc-47::GFP (DD and VD neurons, green) together with Pflp-13::mCherry (DD neurons, red). Commissures with both red and green fluorescence (yellow) are DD neurons, while VD commissures are labeled solely by green fluorescence. (A) Wild type commissures of each neuron class fully extend from the ventral to the dorsal nerve cord. Misguided VD (B) and DD (C) commissures are observed in ina-1(gm144) mutants. White arrows indicate prematurely bifurcating “T” shaped commissures. White arrowheads indicate DD commissures. Scale bar, 20 µm. Note that defects in GABAergic commissures often produce gaps or thinning within the dorsal nerve cord (B,C). (D) VD and DD commissures are similarly affected by mutation of ina-1. Error bars indicate standard error of the proportion.