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The C. elegans POU-domain transcription factor UNC-86 regulates the tph-1 tryptophan hydroxylase gene and neurite outgrowth in specific serotonergic neurons

¹ Department of Anatomy and Neurobiology, College of Medicine, University of California, Irvine, Irvine, CA 92697, USA
² Department of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School, Boston, MA 02114, USA

View larger version (94K): [in a new window]   Fig. 1. Effects of unc-86-null mutations on the expression of tph-1::gfp. (A) The position of serotonergic neurons in the head and the axon from HSN (shown in red). The drawing is adapted from Starich et al. (Starich et al., 1995) and White et al. (White et al., 1986). (B) GFP expression of the integrated tph-1-ABCDE::gfp fusion gene in wild-type and unc-86-null adult hermaphrodites. Anterior is towards the left. In wild type, GFP was predominantly expressed in ADF, NSM and HSN. In unc-86-null mutants, GFP expression was observed in ADF, but not in NSM and HSN at any developmental stage. The UNC-86 protein is expressed in NSM and HSN, but not in ADF (Finney and Ruvkun, 1990). Two unc-86-null alleles, n846 and e1416 exhibited the same expression pattern; unc-86(n846) animals are shown. Note that the GFP fusion to the tph-1 sequence has punctate expression pattern, as it appears as two puncta in the ADF neuron.

View larger version (109K): [in a new window]   Fig. 2. unc-86 affects specific genes in the serotonergic neurons. (A-F) Effects of unc-86 on the expression of the cat-1 monoamine vesicular transporter. Expression of an integrated cat-1::gfp fusion gene was examined in wild type, unc-86-null and tph-1 mutants. (A,B) In wild-type, this functional cat-1 fusion to GFP was specifically expressed in all predicted serotonergic and dopaminergic neurons; representative expression in the serotonergic NSM, HSN, dopaminergic PDE neurons and the nerve ring is shown. (C,D) In unc-86 mutants, GFP expression in HSN was completely absent, and in NSM was significantly reduced. However, unlike tph-1::gfp (Fig. 1), cat-1::gfp expression in NSM was not completely absent (C). Note the unc-86 mutants had extra PDE neurons (D) (Finney and Ruvkun, 1990) expressing cat-1::gfp. (E,F) In the serotonin synthesis mutant tph-1(mg280), cat-1::gfp levels in NSM and other neurons were indistinguishable from wild-type. (G,H) Expression of a GFP fusion to the F32G8.6 GTP-cyclohydrolase 1, gtpch-1::gfp, in wild-type (G) and unc-86 mutant (H) animals. In wild-type animals, GFP was observed in all predicted serotonergic and dopaminergic neurons, as well as in the head and body wall muscles (arrowhead). Except for the absence of GFP in HSN (arrowhead in H), the expression levels in unc-86 mutants were very similar to wild type. All animals shown are adults, and the anterior is to the left.

View larger version (123K): [in a new window]   Fig. 3. Analysis of NSM serotonin immunoreactivity in adult hermaphrodites. (A,B) In wild-type animals, nine neurons of five classes were stained by the anti-serotonin antibody. In general, the frequency of the AIM and RIH neurons stained is lower than of NSM, ADF and HSN, and the intensity of the staining in AIM and RIH is often lower. (C,D) In unc-86-null mutants, serotonin immunoreactivity in NSM and ADF remained, unlike in AIM, RIH and HSN. The ADF neurons are the only class of the serotonergic neurons that does not express unc-86, and unc-86 mutations had no effect on ADF serotonin immunoreactivity. (E,F) Treatment of animals with the serotonin reuptake block drug imipramine significantly reduced NSM but not ADF serotonin immunoreactivity in unc-86 mutants (E), and this effect was not observed in wild-type animals treated with imipramine at the same concentration (F). (G) No serotonin could be detected in the unc-86(e1416);nss-1(yz12) double mutant. unc-86(n846); nss-1(yz12) showed similar staining pattern (not shown). (H) The nss-1(yz12) mutation specifically affects serotonin immunoreactivity in ADF. Adult animals are shown, and the anterior is towards the left. For each strain, the immunostaining was performed many times, and quantification of the observation from two recent trials is presented in Table 1.

View larger version (21K): [in a new window]   Fig. 4. tph-1 regulatory elements. The expression of various GFP fusion to tph-1 in transgenic animals. (A) The genomic structure of the tph-1 gene. tph-1 exons are indicated by filled boxes and untranslated regions are indicated as lines. (B) The strength of GFP expression of various constructs. The construct tph-1-ABCDE::gfp was integrated onto the chromosome; different lines have the construct integrated onto different chromosomes. The other constructs were transmitted as extrachromosomal arrays. The number of transgenic lines carrying each construct has been examined: tph-1::ABCDE::gfp, two lines; tph-1-BCDE, four lines; tph-1-CDE::gfp, three lines; tph-1-DE::gfp, four lines; tph-1-E::gfp, three lines; tph-1-BCE::gfp, three lines. Because GFP levels in AIM and RIH are too weak to survey reliably, only GFP expression in ADF, NSM and HSN was scored. GFP expression of tph-1-ABCDE::gfp, tph-1-BCDE::gfp and tph-1-CDE::gfp in HSN was observed only in adults, tph-1-DE::gfp was expressed in HSN starting at the first larval stage.

View larger version (106K): [in a new window]   Fig. 5. Analysis of NSM morphology in unc-86 mutants. Serotonergic neurons in wild-type (A) and unc-86 mutants (B-E) were visualized by anti-serotonin antibody staining. In wild-type animals (A), NSM has two posterior-directed processes. The position and the gross axon morphology of NSM in unc-86-null mutants are indistinguishable from wild-type, but neurite outgrowth defects were observed. Representative examples of outgrowth defects of NSM are shown. The arrows indicate neurite outgrowth defects in the corresponding neurons. In most cases, an extra neurite extended anteriorly to various lengths (B), one of the posteriorly directed axon was either premature terminated (C) or missing (D). In some cases, NSM had one anterior- and one posterior-directed process (E). Similar defects were observed in unc-86(n846) and unc-86(e1416), and there was no obvious difference in the frequency of the defects observed in the larvae and adults.

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