Lysophospholipid receptors LPA1-3 are not required for the inhibitory effects of LPA on mouse retinal growth cones

Abstract

One of the major requirements in the development of the visual system is axonal guidance of retinal ganglion cells toward correct targets in the brain. A novel class of extracellular lipid signaling molecules, lysophospholipids, may serve as potential axon guidance cues. They signal through cognate G protein-coupled receptors, at least some of which are expressed in the visual system. Here we show that in the mouse visual system, a lysophospholipid known as lysophosphatidic acid (LPA) is inhibitory to retinal neurites in vitro when delivered extracellularly, causing growth cone collapse and neurite retraction. This inhibitory effect of LPA is both active in the nanomolar range and specific compared to the related lysophospholipid, sphingosine 1-phosphate (S1P). Knockout mice lacking three of the five known LPA receptors, LPA_1-3, continue to display retinal growth cone collapse and neurite retraction in response to LPA, demonstrating that these three receptors are not required for these inhibitory effects and indicating the existence of one or more functional LPA receptors expressed on mouse retinal neurites that can mediate neurite retraction.

Keywords: axon guidance; lysophosphatidic acid; retinal ganglion cells.

Figure 1

Collapse of mouse retinal growth cones after treatment with LPA. Fluorescent images of F-actin stained with TRITC-Phalloidin from two examples of growth cones under control conditions (a) or two examples of collapsed retinal growth cones after treatment with 100 nM LPA for 30 minutes (b). Note abundant lamellipodia and filopodia under control conditions (a) which are absent in collapsed growth cones treated with LPA (b). Scale bar: 10 μm. (c) Graph of quantification of growth cone collapse (100% indicating all growth cones collapsed) assayed after 30 minutes of treatment with various concentrations (log scale, molar) of LPA (●) shows a dose-dependent response compared to control (▲) or S1P treatment (♦). Data from multiple explants of multiple experiments (see Supplementary Table 1); bars show SEM. Abbreviations: LPA, lysophosphatidic acid; SEM, standard error of mean.

Figure 2

LPA-induced growth cone collapse in Lpar2 mutant retina neurites. Growth cones from E14.5 retinal neurites under control conditions (a–c) or after treatment with 100 nM LPA for 30 minutes (d–h) stained with TRITC-Phalloidin. Retinal growth cones from Lpar2 mutant embryos (b–c, f–h) show similar morphology to wild type littermates (a, d–e), both under control conditions (a–c) and after LPA treatment (d–h). Similar growth cone collapse is seen in the Lpar2 mutant retina as in wild type. Scale bars: 15 μm. Abbreviation: LPA, lysophosphatidic acid.

Figure 3

Quantification of growth cone collapse of retinal neurites from LPA receptor mutants. Percentage of growth cones showing collapsed morphology from observations of retinal explants of various mouse LPA receptor genotypes under control conditions (0 LPA) or 1 nM, 10 nM, or 100 nM LPA concentrations (log scale, molar). Each data point is from multiple explants collected from multiple animals (see Supplementary Table 2), with bars representing SEM from different animals. Wild type data are from littermates. Abbreviations: LPA, lysophosphatidic acid; SEM, standard error of mean.

Figure 4

Time-lapse images of retinal growth cone response to LPA. DIC images from time-lapse recordings of a retinal growth cone growing in culture on laminin. Sequence of images show growth from 30 minutes prior to LPA treatment (t = −30) to time of addition of LPA (t = 0). Subsequently, 2.5 minutes after LPA addition (t = 2.5), the growth cone has begun to collapse (arrow), and by 3 minutes after LPA addition (t = 3), the growth cone has completely collapsed and the neurite is retracting. A small piece of debris (arrowhead) is indicated for reference. Time in minutes is designated relative to time of LPA addition (t = 0). Abbreviations: LPA, lysophosphatidic acid; SEM, standard error of mean; t, time; DIC, differential interference contrast.

Figure 5

Neurite retraction after LPA treatment. Phase contrast image montages of retinal explants and neurites from wild type E14.5 mouse embryos (a–b) and Lpar1 Lpar2 Lpar3 triple mutant embryos (c–d). The same explants were imaged before (a, c) and after (b, d) LPA treatment for 30 minutes, showing dramatic neurite retraction. Scale bars: 200 μm. Abbreviation: LPA, lysophosphatidic acid.

Figure 6

Quantification of neurite retraction. Neurite retraction from wild type mouse retinal explants shows a dose-dependent response to LPA (a). Neurite retraction is measured as percent of total neurite length lost per explant after 30 minute incubation with 1 nM, 10 nM, or 100 nM LPA (log scale, molar) or control media (0) compared to total neurite length before treatment. 100% indicates complete loss of neurites, 0% retraction indicates no net change, and negative values indicate growth. Neurite retraction was observed in retinal explants from LPA receptor mutants after treatment with 10 nM LPA for 30 minutes (b). No significant differences in neurite retraction levels were observed between mutant and wild type retinal explants (ANOVA). Data presented from multiple explants of multiple experiments (see Supplementary Table 3); bars show SEM. Abbreviations: LPA, lysophosphatidic acid; ANOVA, analysis of variation; SEM, standard error of mean.