A gene expression fingerprint of C. elegans embryonic motor neurons

Abstract

Background: Differential gene expression specifies the highly diverse cell types that constitute the nervous system. With its sequenced genome and simple, well-defined neuroanatomy, the nematode C. elegans is a useful model system in which to correlate gene expression with neuron identity. The UNC-4 transcription factor is expressed in thirteen embryonic motor neurons where it specifies axonal morphology and synaptic function. These cells can be marked with an unc-4::GFP reporter transgene. Here we describe a powerful strategy, Micro-Array Profiling of C. elegans cells (MAPCeL), and confirm that this approach provides a comprehensive gene expression profile of unc-4::GFP motor neurons in vivo.

Results: Fluorescence Activated Cell Sorting (FACS) was used to isolate unc-4::GFP neurons from primary cultures of C. elegans embryonic cells. Microarray experiments detected 6,217 unique transcripts of which approximately 1,000 are enriched in unc-4::GFP neurons relative to the average nematode embryonic cell. The reliability of these data was validated by the detection of known cell-specific transcripts and by expression in UNC-4 motor neurons of GFP reporters derived from the enriched data set. In addition to genes involved in neurotransmitter packaging and release, the microarray data include transcripts for receptors to a remarkably wide variety of signaling molecules. The added presence of a robust array of G-protein pathway components is indicative of complex and highly integrated mechanisms for modulating motor neuron activity. Over half of the enriched genes (537) have human homologs, a finding that could reflect substantial overlap with the gene expression repertoire of mammalian motor neurons.

Conclusion: We have described a microarray-based method, MAPCeL, for profiling gene expression in specific C. elegans motor neurons and provide evidence that this approach can reveal candidate genes for key roles in the differentiation and function of these cells. These methods can now be applied to generate a gene expression map of the C. elegans nervous system.

Figure 1

MAPCeL strategy for profiling C. elegans GFP neurons. Embryos are isolated from gravid adults and treated with chitinase to degrade the egg shell. Embryonic cells are cultured for 24 hours and enriched by FACS. Amplified, labeled aRNA is hybridized to the Affymetrix C. elegans array.

Figure 2

Isolation of unc-4::GFP neurons by FACS. A. Fluorescence intensity profile of wildtype (non-GFP) cells. Boxed areas exclude autofluorescent7 cells (arrow). B. unc-4::GFP cells are gated to exclude propidium iodide-stained (non-viable) cells. C. Light scattering gate for GFP-positive cells (circle) to exclude cell clumps and debris. D. Combined fluorescence and DIC image of unc-4::GFP labeled motor neurons in L1 larva. (DA2 is not visible here.) Arrow points to embryo at stage (< 400 min) prior to unc-4::GFP expression. E, F. Fluorescence and DIC images of 24 hr culture from unc-4::GFP embryos. G. unc-4::GFP neurons after enrichment by FACS. Arrow heads point to rare (~10%) non-GFP cells. Scale bars are 5 microns.

Figure 3

Coefficients of Determination (R²) for individual hybridizations. A. Scatter plot of normalized intensity values (log base 2) for representative hybridization (DMR30) from all cells (Reference) compared to the average intensity of four Reference hybridizations. B. Scatter plot of representative unc-4::GFP hybridization (DM39) compared to the average intensities for all three unc-4::GFP hybridizations. C. Results of single unc-4::GFP hybridization (DM39) (red) compared to average Reference intensities (green) to identify transcripts showing differential expression. The unc-4 transcript (arrowhead) is highly enriched (~13×) in unc-4::GFP neurons D. R² values for all pairwise combinations of unc-4::GFP hybridizations. E. R²values for all pairwise combinations of Reference (i.e. all cells) hybridizations.

Figure 4

Comparison of Expressed Genes (EGs) in unc-4::GFP neurons vs all cells (N2 reference). 968 transcripts are detected exclusively in unc-4::GFP motor neurons and 3854 transcripts are detected exclusively in the N2 reference data set. 5249 transcripts are detected in both data sets.

Figure 5

GFP reporters validate UNC-4 motor neuron genes. A. unc-4::GFP is expressed in 13 embryonically-derived motor neurons. Colored circles indicate approximate location of UNC-4 motor neuron soma in newly hatched L1 larva. Transgenic animals expressing GFP reporters for representative UNC-4 motor neuron genes. Anterior to left. B. acr-14. Confocal GFP/DIC projection. acr-14::GFP is expressed in RME in the head and in ventral nerve cord (VNC) motor neurons (arrows). C, D. flp-13. Confocal DIC/GFP image of head region. (C) and matched confocal GFP projection (D). Note DD1 commissure to dorsal side (white arrow). Arrowhead marks nerve ring. E, F. F29G6.2. Arrows point to VNC motor neurons (L2 Larva) (E). Posterior view of VNC showing F29G6.2::GFP expression in all VNC motor neurons. Asterisk marks gut autofluorescence (F). G, H. tig-2. DIC/GFP image of L2 larva. Note GFP expression in VNC motor neurons (arrows) and in head muscles (arrowhead) (G). Confocal projection of anterior VNC. tig-2::GFP is detected in A and B class motor neurons, pharyngeal muscle (arrow) and body wall muscle (bwm) (H). Scale bars are 10 microns in panels C, D, F, H and 20 microns in B, E, G.

Figure 6

Model of DA motor neuron axon guidance. Ventrally derived UNC-6/Netrin guidance cues binds to the UNC-40/DCC and UNC-5 receptor to steer the DA motor axon toward the dorsal nerve cord. The receptor tyrosine phosphatase, CLR-1, promotes dorsal motor axon outgrowth via an UNC-40/DCC independent pathway [24]. The transcript encoding the C. elegans ortholog of Abelson tyrosine kinase (ABL-1) is enriched in the unc-4::GFP motor neuron data set and is proposed to antagonize CLR-1 activity.

Figure 7

G-protein signaling pathways regulating neurotransmitter release in cholinergic motor neurons. Components shown in bold are enriched in the unc-4::GFP motor neuron data set. All others are EGs with the exception of the dop-3 transcript which is not detected (light gray text). See Table 3 for protein descriptions. Green denotes interactions that promote acetylcholine (ACh) release and red marks steps that inhibit synaptic vesicle fusion. Neurotransmitters are highlighted in gray boxes. This figure adapted from Reynolds et al. (2004) [60].

Figure 8

Signaling components detected in unc-4::GFP motor neurons.