Transcriptional regulation and stabilization of left-right neuronal identity in C. elegans

Abstract

At discrete points in development, transient signals are transformed into long-lasting cell fates. For example, the asymmetric identities of two Caenorhabditis elegans olfactory neurons called AWC(ON) and AWC(OFF) are specified by an embryonic signaling pathway, but maintained throughout the life of an animal. Here we show that the DNA-binding protein NSY-7 acts to convert a transient, partially differentiated state into a stable AWC(ON) identity. Expression of an AWC(ON) marker is initiated in nsy-7 loss-of-function mutants, but subsequently lost, so that most adult animals have two AWC(OFF) neurons and no AWC(ON) neurons. nsy-7 encodes a protein with distant similarity to a homeodomain. It is expressed in AWC(ON), and is an early transcriptional target of the embryonic signaling pathway that specifies AWC(ON) and AWC(OFF); its expression anticipates future AWC asymmetry. The NSY-7 protein binds a specific optimal DNA sequence that was identified through a complete biochemical survey of 8-mer DNA sequences. This sequence is present in the promoter of an AWC(OFF) marker and essential for its asymmetric expression. An 11-base-pair (bp) sequence required for AWC(OFF) expression has two activities: One region activates expression in both AWCs, and the overlapping NSY-7-binding site inhibits expression in AWC(ON). Our results suggest that NSY-7 responds to transient embryonic signaling by repressing AWC(OFF) genes in AWC(ON), thus acting as a transcriptional selector for a randomly specified neuronal identity.

Figure 1.

nsy-7(ky630) mutants are defective for maintenance of asymmetric identity in AWC. (A) Adult animal with the integrated array kyIs408. A fluorescence image of AWC^ON, expressing str-2∷dsRed2, and AWC^OFF, expressing srsx-3∷GFP, is overlaid on a DIC image. (B) AWC neurons in wild-type animals in early L1 (1 h after hatching), late L1 (14 h after hatching), or in the young adult. (Red fluorescence) str-2∷dsRed2; (green fluorescence) srsx-3∷GFP. (C) Quantitation of srsx-3∷GFP and str-2∷dsRed fluorescence (n = 25–38 animals). In each animal, the cell with higher srsx-3∷GFP expression was defined as AWC^OFF. (D) Fraction of nsy-7(ky630) animals expressing str-2∷dsRed2 at 1, 5, 14, and 70 h after hatching; 1–14 h are L1 stage, and 70 h is adult. (E) AWC neurons in nsy-7(ky630) animals, as in B. (F) Quantitation of srsx-3∷GFP and str-2∷dsRed fluorescence in nsy-7(ky630) mutants, as in C (n = 33–45 animals).

Figure 2.

nsy-7 encodes a protein with distant similarity to homeodomains. (A) Sequence of the long isoform of NSY-7. The ky630 and tm3080 mutations are marked, and the predicted homeodomain-like region is underlined. Dotted line indicates residues absent in the short NSY-7 isoform. (B) Alignment of the NSY-7 homeodomain-like region with engrailed-family homeodomains. Engrailed residues that bind specific bases (open circles) or the DNA backbone (black circles) and residues that form the hydrophobic core (gray circles) are marked. Asterisks above the sequences mark residues important for homeodomain function (Gehring et al. 1994; Draganescu and Tullius 1998; Fraenkel et al. 1998; Sato et al. 2004; Chi 2005). (C) Expression of srsx-3∷GFP and str-2∷dsRed in AWC neurons of wild type, nsy-7 mutants, C18F3.4-RNAi animals, and nsy-7(tm3080) rescued with a genomic clone or an odr-3∷nsy-7 cDNA clone. Numbers of animals scored are indicated. (D) Percentage of nsy-7(tm3080) animals expressing str-2∷dsRed2 at 1, 5, 14, and 70 h after hatching; 1–14 h are L1 stage, and 70 h is young adult. (E) Chemotaxis to the AWC^ON-specific odor 2-butanone and the AWC^OFF-specific odor 2,3-pentanedione.

Figure 3.

Expression of nsy-7∷GFP and nuclear localization of NSY-7. (A) Expression of a 21-kb nsy-7∷GFP promoter fusion (green) and the AWC marker odr-1∷mCherry (red). The AWC cell body and AWC axons in the nerve ring are marked. (B) nsy-7∷GFP expression across development. (C) Nuclear localization of a NSY-7∷GFP protein expressed in both AWCs under the odr-3 promoter. str-2∷dsRed marks AWC^ON; note gain-of function 2-AWC^ON phenotype. (D) Cytoplasmic localization of NSY-7rGFP with the ky630 mutation (odr-3 promoter, str-2∷dsRed marker); note wild-type asymmetric expression of str-2∷dsRed. (E) Quantification of str-2∷dsRed expression in wild type, or wild type overexpressing odr-3∷nsy-7∷GFP with or without the ky630 mutation. Numbers of animals scored are indicated.

Figure 4.

NSY-7 binds to the sequence CCTTAAC. (A) Consensus NSY-7-binding sequence determined by protein-binding microarray experiments. The enrichment score correlates with the binding affinity of NSY-7 for the sequence, and is measured on a scale of −0.5 to 0.5. (B) In vitro binding of 6His-tagged NSY-7 to the predicted consensus sequence. FL, full-length consensus sequence in a 39-nucleotide context from the srsx-3 promoter. Consensus binding site is boxed. M1 and M2, mutations predicted to decrease the enrichment score (E.S.), shown to the right of the sequences. All lanes except the first contain 500 ng of 6His:NSY-7. Lanes 5–9 contain a labeled FL probe and increasing amounts of cold FL competitor; lanes 10–14 show labeled FL probe + cold M2. The bottom band in lanes 2–4 probably represents a degradation product of the recombinant protein. (C,D) Mutations made in the srsx-3 promoter (C), and resulting expression patterns (D). Enrichment scores are shown for each point mutation. (+++) 80%–100% of population; (++) 30%–80%; (+) 10%–30%; (blank) <1%. (E) GFP expression under an srsx-3 promoter containing mutations 7 and 8 (0 AWC). (F) GFP expression under an srsx-3 promoter containing mutation 11 (1 AWC). (G) GFP expression under an srsx-3 promoter containing mutations 9 and 10 (2 AWC). (H) DIC image of worm shown in G. In E–H, asterisks indicate AWB and arrowheads indicate AWC.

Figure 5.

hlh-11 is regulated by nsy-7. (A) Expression of an hlh-11∷GFP reporter with 3 kb of sequence upstream of the start site, showing colocalization with the AWC marker odr-1∷mCherry. (B) hlh-11∷GFP expression in wild-type, nsy-1(ky542), and nsy-7(tm3080) mutant backgrounds. Asterisk indicates results different at P < 0.001 (χ² test). Numbers of animals scored are indicated.

Figure 6.

Developmental stabilization of stochastic AWC asymmetry. Gene expression patterns in AWC^ON and AWC^OFF across development are shown, along with inferred roles of the initial signaling cascade, the nsy-7 transcriptional regulator, and the odr-1/egl-4 activity-dependent pathway.