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First published online March 24, 2005
doi: 10.1242/10.1242/dev.01788

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Regulation of chemosensory and GABAergic motor neuron development by the C. elegans Aristaless/Arx homolog alr-1

Department of Biology, Brandeis University, 415 South Street, Brandeis, MA 02454, USA

View larger version (21K): [in a new window]   Fig. 1. alr-1 encodes the C. elegans homolog of Aristaless/ARX. (A) Phylogenetic tree of the homeodomains of Aristaless/ARX family members. The homeodomain sequences were aligned and analyzed using Bonsai 2.1 (http://calliope.gs.washington.edu/software/index.html). The homeodomains of OTD/OTX proteins were used as outliers in this analysis. The frequency at which a particular branch appears in 1000 bootstrap replicates are indicated by hatchmarks: (/) – <50%; (//) – 51-80%; (///) – 81-100%. (B) Alignment of the homeodomains of ALR-1 (Ce), AL (Dm) and ARX (Mm, Hs). Identical residues are indicated in gray. The Q50 residue is boxed. (C) (Top) alr-1 and neighboring predicted open reading frames present on the cosmid R08B4. The orientation of each ORF is indicated by an arrow. Sequences deleted in alr-1(oy42) are indicated by a dashed line; inverted sequences are indicated by a dotted line. (Bottom) Exon/intron structure of alr-1. Sequences encoding the homeodomain are hatched. Sequences deleted in alr-1(ok545) are indicated by a dashed line. Asterisk indicates a point mutation in the first splice donor site in alr-1(oy56). (D) Drosophila Al can functionally substitute for alr-1 in the regulation of odr-7::gfp and ops-1::dsRed expression, and for the dye-filling phenotype. The Al cDNA was expressed under the alr-1 promoter. Adult animals grown at 25°C were examined. n>100 for each.

View larger version (113K): [in a new window]   Fig. 2. Mutations in alr-1 affect gene expression and morphology of the AWA olfactory neurons. (A-D) Top-down views of adult wild type (A), or alr-1(oy42) (B-D) animals stained with anti-ODR-7 antibodies. Nuclei of the AWA neurons are indicated by arrowheads. alr-1(oy42) mutants exhibit loss of ODR-7 expression in one (B) or both (C) AWA neurons, or ectopic ODR-7 expression (D). (E,F) Lateral view of merged Nomarski and fluorescent images of a wild-type (E) or alr-1(oy42) animal (F) expressing an odr-10::gfp fusion gene that allows visualization of the AWA sensory dendrite. Arrows indicate dendritic termination points. Scale bar: 10 µm.

View larger version (18K): [in a new window]   Fig. 3. alr-1 mutants fail to respond to an AWA-sensed odorant. All strains except odr-3(n2150) contain stably integrated copies of odr-7::gfp. The responses of single animals of the indicated genotypes to a point source of a 1:1000 dilution of diacetyl or 1:200 dilution of benzaldehyde were examined. alr-1(oy42) and alr-1(oy56) animals expressed odr-7::gfp in both (1/1) or neither (0/0) AWA neuron(s). Asterisks indicate responses that are significantly different from wild-type responses (P<0.001). #, responses of animals lacking odr-7::gfp expression in both AWA neurons that are significantly different from the responses of animals of the same genotype expressing odr-7::gfp in both AWA neurons at P<0.001; nx30 for each.

View larger version (18K): [in a new window]   Table 2.

View larger version (51K): [in a new window]   Fig. 4. Expression pattern of alr-1. (A-D) Embryonic expression of ALR-1. (A) Nomarski image and (B) anti-ALR-1 antibody staining of a 1.5-fold embryo. ALR-1 expression is observed in two cells in the anterior, only one of which is visible in this plane of focus. (C) Nomarski and (D) anti-ALR-1 antibody stained threefold embryo exhibiting ALR-1 expression in multiple cells in the head, body and tail. Shown is a merge of three different focal planes. (E-J) Post-embryonic expression of ALR-1. Adult animals expressing the GABAergic neuronal marker unc-47::gfp in the head (E,F) or tail (G,H) were co-stained with anti-ALR-1 antibodies. Identified cells co-expressing both markers are labeled. Ventral cord motoneurons expressing both markers are indicated by arrowheads. Additional ALR-1-expressing cells were not identified. (I,J) Adult animals stained with anti-ODR-7 antibodies (I) or expressing ops-1::gfp (J) were stained with anti-ALR-1 antibodies. ODR-7-expressing AWA neurons and ops-1::gfp-expressing ASG neurons are indicated by arrows. No ALR-1 expression was observed in these neurons. Scale bar: 10 µm.

View larger version (18K): [in a new window]   Fig. 5. ALR-1 may act in the AWA/ASG lineage to regulate neuronal development. (A) Part of the described embryonic lineage giving rise to the AWA, ASI and ASK neurons on the left and right sides of an animal (Sulston et al., 1983). (B) Presence or absence of odr-10::gfp expression in an AWA neuron on a side is more closely correlated with presence or absence of an extrachromosomal array containing alr-1 rescuing sequences and osm-6::dsRed in the ASI than in the ASK neurons in alr-1(oy42) animals. * indicates significantly different from ASI at P<0.05 by X-square analysis. n=44 each for odr-10::gfp+ and odr-10::gfp– neurons. L1-L3 larvae grown at 25°C were examined (see Materials and methods).

View larger version (92K): [in a new window]   Fig. 6. alr-1 mutants exhibit ectopic flp-13::gfp expression. (A-F) flp-13::gfp expression in adult animals. Ventral cord motoneurons expressing flp-13::gfp in wild-type (B), alr-1(oy42) (D) and unc-55 (e402) (F) animals are indicated by arrowheads. Corresponding Nomarski images are shown on the left (A,C,E). (G-I) Merged Nomarski and fluorescent images of flp-13::gfp expression in wild-type (G), alr-1(oy42) (H) and unc-55(e402) (I) L1 larvae. GFP-expressing ventral cord motoneurons are indicated by arrowheads. Scale bar: 50 µm.

View larger version (69K): [in a new window]   Fig. 7. Ectopic flp-13::gfp-expressing cells in alr-1 mutants are GABAergic. Colocalization (D,H) of flp-13::gfp expression (C,G) with anti-GABA staining (B,F) in ventral cord motoneurons of wild-type (A-D) or alr-1(oy42) mutants (E-H). Scale bar: 10 µm.

View larger version (15K): [in a new window]   Fig. 8. Model for ALR-1 functions in the AWA/ASG sensory and VD/DD MNs. (A) ALR-1 acts in parallel to UNC-130 and upstream of LIN-11 to specify the AWA and ASG neurons. ALR-1 may act in the precursors or earlier in the lineage. (B) ALR-1 and UNC-55 act in parallel to regulate flp-13::gfp expression in the VD MNs. The function of ALR-1 in the DD MNs is unclear. UNC-55 also acts to specify the correct synaptic connectivities of the VD MNs. Expression of the unc-25 GAD and unc-47 vesicular GABA transporter genes is regulated by the UNC-30 HD protein (see text for references).

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