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First published online 3 August 2006
doi: 10.1242/dev.02494

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An unusual Zn-finger/FH2 domain protein controls a left/right asymmetric neuronal fate decision in C. elegans

¹ Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center, 701 W. 168th Street, New York, NY 10032, USA.
² Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada.
³ Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Center for Computational Biology and Bioinformatics, Columbia University Medical Center, 1130 St Nicholas Avenue, Room 815, New York, NY 10032, USA.
⁴ Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.

View larger version (43K): [in a new window]   Fig. 1. fozi-1 mutants display a de-repression of ASEL-specific fate markers in ASER. (A) Summary of previously described regulatory interactions that determine ASEL and ASER fate. Some permissively acting genes (Chang et al., 2003) are not shown for simplicity. (B) In fozi-1(ot61) mutants, ASEL-specific gcy-7prom::gfp (otIs3) and lim-6prom::gfp (otIs114) expression is de-repressed in ASER. See Table 1 and Fig. 4A for quantification.

View larger version (17K): [in a new window]   Fig. 2. fozi-1 encodes a protein with two Zn fingers and a formin homology 2 domain. (A) Mapping of the ot61 mutation. (B) Structure of the predicted FOZI-1 protein.

View larger version (39K): [in a new window]   Fig. 3. Rescue, expression, site of action and domain requirements of fozi-1. (A) Rescue of the fozi-1 mutant phenotype. The constructs do not contain the first, non-coding exon of the fozi-1 gene, which is located >7 kb upstream of the ATG-containing second exon (Fig. 2A). The right column indicates rescue of the fozi-1 defect, i.e. suppression of aberrant lim-6 expression in ASER, and the left column indicates suppression of normal lim-6 expression in ASEL fate, caused by ectopic expression of fozi-1. All constructs show similar expression levels and exclusive localization to the nucleus. (B) A representative fozi-1::gfp-expressing animal, showing gfp expression in ASER but not ASEL, and in the two olfactory neurons AWCL and AWCR. Broken lines approximately indicate the head of the worm. The insets show that fozi-1::gfp predominantly localizes to the nucleus. The red `cyto' marker is dsRed2 protein, expressed under control of the ceh-36 promoter (otIs151 transgene). (C) Three out of four fozi-1::gfp-expressing transgenic, wild-type lines display varying levels of asymmetric gfp expression in ASER. Circles indicate absent, ASEL alone, ASEL and ASER, and ASER alone, respectively.

View larger version (42K): [in a new window]   Fig. 4. Analysis of ASEL and ASER fate in fozi-1 and lim-6 mutant animals. (A) In fozi-1(cc607)-null mutant animals, ASEL-specific lim-6prom::gfp (otIs114), flp-4prom::gfp (otIs178), gcy-7 prom::gfp (otIs3) and gcy-6 prom::gfp (otIs162) are de-repressed in ASER. `0=0' indicates no expression, `L>0' indicates exclusive expression in ASEL, `L>R' indicates expression in ASEL is stronger than in ASER, `L=R' indicates equal expression in ASEL and ASER, `L<R' indicates expression in ASER is stronger than in ASEL, and `0<R' indicates exclusive expression in ASER. (B) Analysis of ASER fate markers in fozi-1(cc607) null mutants. Reporter arrays used were ntIs1 (gcy-5), otEx2409 (gcy-4) and otEx1274 (hen-1ASER). (C) Summary of the fozi-1 mutant phenotype and comparison with previously described ASE fate mutants. Blue circles indicate the ASEL-specific gene expression battery, red circles indicate the ASER-specific gene expression battery. (D) lim-6 is not sufficient to repress ASER cell fate. (E) Summary of genetic interaction data. The incomplete expressivity of fozi-1 and lim-6 null alleles argues for the existence of parallel pathways (indicated by factor X, Y, Z). The two different scenarios make different predictions about the site of action of the parallel pathways. In scenario 1, it is active in ASER; in scenario 2, it is active in ASEL.

View larger version (20K): [in a new window]   Fig. 5. fozi-1 acts downstream of the bi-stable feedback loop to control ASER differentiation. (A) Asymmetric fozi-1::gfp expression (otEx2192; line #3 in Fig. 3C) is disrupted in die-1(ot26), lsy-6(ot71) and cog-1(sy607) null mutants animals, and partially affected in lim-6-null mutants. The `L=R' category indicates equal expression in ASEL and ASER, including de-repression of gfp expression in ASEL (yielding strong gfp expression in both ASEL and ASER; die-1 and lsy-6 phenotype) and reduction of gfp expression in ASER (yielding equally low expression in ASEL and ASER; cog-1 phenotype). (B) Removing fozi-1 reverts the loss of lim-6 expression (otIs114) observed in lsy-6(ot71) or die-1(ot26) mutant animals. (C) Summary of genetic interactions, pooling data from A and B, combined with the data from Fig. 4. The completely penetrant and expressive phenotype observed in die-1 mutants argues that the pathways parallel to fozi-1 and lim-6 are under die-1 control. The simplest explanation is that die-1 itself acts in parallel to fozi-1 and lim-6 to control expression of target genes. (D) fozi-1(cc607) null mutant animals display weakly penetrant defects in lsy-6 expression, assayed with lsy-6prom::gfp (otIs162) and cog-1 expression, assayed with cog-1::gfp (syIs63). (E) Summary of the feedback data. The lim-6- dependent feedback to lsy-6 (or die-1; for simplicity, the arrow only points to lsy-6), described in Johnston et al. (Johnston et al., 2005), is represented by a broken arrow to indicate that the effect is partially penetrant and only required to maintain the asymmetric expression of loop components.

View larger version (60K): [in a new window]   Fig. 6. The FH2 domain of FOZI-1 is unusual and does not affect actin dynamics. (A) Alignment of FH2FOZI-1 with other FH2 domains, calculated with T-coffee version 2.03 (Notredame et al., 2000). FH2FOZI-1 shows 9% to 20% sequence identity to other sequences in the alignment (49 FH2 domains from Pfam; only four shown here), which is in the range of sequence identities between the remaining FH2 domain sequences in the alignment. The sequence identities of FH2FOZI-1 and FH2 domains of known structure, yeast FH2Bni-1 [PDB-id:1y64; (Otomo et al., 2005)] and mouse FH2Dia1 [PDB-id:1v9d; (Shimada et al., 2004)], are 16% and 14%, respectively. The profile-profile alignment program hmap (Tang et al., 2003) assigns a very good E-value (1.4e-26) for alignments between FH2FOZI-1 and yeast FH2Bni1. Secondary structure prediction for FH2FOZI-1 also agrees well with the secondary structure found in the known structures (colored bar above alignment for FH2Bni1). Neither Ile1431 nor Lys1601, which are crucial for the actin nucleation activity of the FH2 domains are conserved in FH2FOZI-1 (red and black arrow). The colored bar at the bottom shows sequence conservation as calculated previously (Valdar, 2002) (red, high degree of conservation; blue, low degree of conservation). (B) Dimeric structure of the FH2 domain of yeast Bni1 (Otomo et al., 2005). (C) FH2FOZI-1 does not stimulate actin polymerization. The FH2 domain of mouse Dia1 serves as a positive control. (D) FH2FOZI-1 multimerizes. Isolated FH2FOZI-1 (predicted to be 42 kDa) migrates as a single band (lane 1, -BMH). Treatment of FH2FOZI-1 with the crosslinking reagent Bis-maleimidohexane (BMH) produces discrete slower migrating bands (lane 3, +BMH). The putative FH2 multimer is not present if the protein sample is denatured prior to crosslinking (lane 2, +SDS+BMH). FH2FOZI-1 was detected by immunoblotting.

View larger version (42K): [in a new window]   Fig. 7. Summary of the gene regulatory architecture in the ASE neurons. (A) Summary of regulatory interaction in ASEL and ASER. Broken line indicates partially penetrant feedback interaction (see Fig. 5E). See D for deconvolution of individual regulatory interactions. Several permissively acting factors, i.e. factors expressed in both ASEL and ASER (Chang et al., 2003) are not shown here for simplicity. Such factors could, for example, activate the ASER-expressed GCY genes in the absence of the ASEL repressors die-1 and lim-6. (B) Network motifs. A FFL motif occurs when one gene (Gene A) controls a second gene (Gene B) and together these factors are required to regulate a target gene (Gene T). The addition of other factors (e.g. factor C) transforms the FFL motif to a `bi-parallel motif' (Milo et al., 2002), which (analagous to FFL motifs) one could also envision to work as a persistence detector. (C) die-1 and fozi-1 may control target genes through a FFL motif. For a target to be activated, it requires both the presence of die-1 and the absence of fozi-1. See D for identity of target genes. All arrows shown in this figure represent genetic interactions and do not necessarily imply direct physical interactions. Therefore, the identification of additional factors may alter network architecture. For example, die-1 may not only repress fozi-1 but also an additional factor, `repressor Z', which together with fozi-1 may repress ASEL-specific GCY genes. Such a repressor Z would transform the network motif from a FFL motif to a `bi-parallel motif' (B). (D) Deconvoluted regulatory motifs extracted from A. Owing to their differential behavior upon loss of upstream regulators, ASE terminal differentiation genes can be placed into three distinct categories, all of which controlled by the basic FFL motif architecture shown in B. Target Gene Category 1: ASEL-specific expression of the GCY genes gcy-6, gcy-7, gcy-14 and gcy-20 does not require lim-6, but depends on the loop output regulator die-1 and the downstream regulator fozi-1. As a complete elimination of fozi-1 activity only results in partially expressive de-repression of the ASEL-specific GCY genes, an additional factor must be involved in repressing these GCY genes. This factor could be an unknown repressor that cooperates with fozi-1 or, alternatively, the failure to completely activate ASEL-specific GCY genes in ASER may be due to the lack of an activator in ASER (Fig. 4E). The loop output regulator die-1 is the best available candidate for this activator as die-1 is predominantly expressed in ASEL and die-1 mutation leads to a completely penetrant and expressive effect on ASEL-specific gene expression. As die-1 also regulates fozi-1, the genetic interaction therefore may define a FFL motif. This motif is the most parsimonious illustration of the genetic observation that ASEL-specific genes depend on two different factors: the presence of die-1 and the absence of fozi-1. Target Gene Categories 2 and 3: Regulation of genes in this category is distinguishable from control of Category 1 genes by the distinct role of the LIM homeobox gene lim-6. die-1 represses fozi-1 expression in ASEL; in the absence of fozi-1, lim-6 is expressed. Together, lim-6 and die-1 (or a die-1-dependent pathway) activate ASEL-specific FLP genes and repress ASER-specific GCY genes in ASEL. This motif architecture is also a FFL motif, but now with an additional tier of regulation. Similar to the case of Category 1 target genes, the argument for this network architecture is revealed through the completely penetrant effect of disruption of the components of the feedback loop (including die-1) on all downstream genes (lim-6, fozi-1 and terminal target genes), and the incompletely penetrant and expressive effect of lim-6 on the terminal target genes. This incomplete penetrance and expressivity implies the need for another regulatory factor, for which die-1 is at present the best candidate, given its completely penetrant and expressive effect on the terminal target genes. An additional potential feed-forward motif in the interaction of these factors is suggested by the incomplete penetrance of fozi-1 on lim-6 expression. As lim-6 is affected by die-1 in a completely penetrant manner, lim-6 is controlled by a potential feed-forward loop, receiving inputs from die-1 and fozi-1. This renders lim-6 under the same control as the above mentioned ASEL-specific GCY genes, which are also controlled by a combination of die-1 and fozi-1.

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