The C. elegans Frizzled CFZ-2 is required for cell migration and interacts with multiple Wnt signaling pathways

AbstractMembers of the Frizzled family of integral membrane proteins are implicated in many developmental events, including specifying cell fate, orienting cell and planar polarity, and directing cell migration. Frizzleds function as cell surface receptors for secreted Wnt proteins. We report here the isolation of a mutation in cfz-2, a Caenorhabditis elegans Frizzled gene. Mutation of cfz-2 causes defective cell migration, disorganization of head neurons, and can cause ectopic axon outgrowth. Analysis of mosaic animals shows that CFZ-2 functions cell nonautonomously, but does not rule out an autonomous role. CFZ-2 is expressed primarily in the anterior of embryos and in several cells in the head of adults. Our analysis of interactions between CFZ-2 and other Wnt pathways reveals that three Wnts, CWN-1, CWN-2 and EGL-20, and a Frizzled, MOM-5, function redundantly with one another and with CFZ-2 for specific cell migrations. In contrast, CWN-1, CWN-2, EGL-20, CFZ-2, and MOM-5 antagonize one another for other migrations. Therefore, CFZ-2 functions by collaborating with and/or antagonizing other Wnt signaling pathways to regulate specific cell migrations

Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal

MicroRNAs are regulators of gene expression whose functions are critical for normal development and physiology. We have previously characterized mutations in a Caenorhabditis elegans microRNA-specific Argonaute ALG-1 (Argonaute-like gene) that are antimorphic [alg-1(anti)]. alg-1(anti) mutants have dramatically stronger microRNA-related phenotypes than animals with a complete loss of ALG-1. ALG-1(anti) miRISC (microRNA induced silencing complex) fails to undergo a functional transition from microRNA processing to target repression. To better understand this transition, we characterized the small RNA and protein populations associated with ALG-1(anti) complexes in vivo. We extensively characterized proteins associated with wild-type and mutant ALG-1 and found that the mutant ALG-1(anti) protein fails to interact with numerous miRISC cofactors, including proteins known to be necessary for target repression. In addition, alg-1(anti) mutants dramatically overaccumulated microRNA* (passenger) strands, and immunoprecipitated ALG-1(anti) complexes contained nonstoichiometric yields of mature microRNA and microRNA* strands, with some microRNA* strands present in the ALG-1(anti) Argonaute far in excess of the corresponding mature microRNAs. We show complex and microRNA-specific defects in microRNA strand selection and microRNA* strand disposal. For certain microRNAs (for example mir-58), microRNA guide strand selection by ALG-1(anti) appeared normal, but microRNA* strand release was inefficient. For other microRNAs (such as mir-2), both the microRNA and microRNA* strands were selected as guide by ALG-1(anti), indicating a defect in normal specificity of the strand choice. Our results suggest that wild-type ALG-1 complexes recognize structural features of particular microRNAs in the context of conducting the strand selection and microRNA* ejection steps of miRISC maturation

The C. elegans histone deacetylase HDA-1 is required for cell migration and axon pathfinding

AbstractHistone proteins play integral roles in chromatin structure and function. Histones are subject to several types of posttranslational modifications, including acetylation, which can produce transcriptional activation. The converse, histone deacetylation, is mediated by histone deacetylases (HDACs) and often is associated with transcriptional silencing. We identified a new mutation, cw2, in the Caenorhabditis elegans hda-1 gene, which encodes a histone deacetylase. Previous studies showed that a mutation in hda-1, e1795, or reduction of hda-1 RNA by RNAi causes defective vulval and gonadal development leading to sterility. The hda-1(cw2) mutation causes defective vulval development and reduced fertility, like hda-1(e1795), albeit with reduced severity. Unlike the previously reported hda-1 mutation, hda-1(cw2) mutants are viable as homozygotes, although many die as embryos or larvae, and are severely uncoordinated. Strikingly, in hda-1(cw2) mutants, axon pathfinding is defective; specific axons often appear to wander randomly or migrate in the wrong direction. In addition, the long range migrations of three neuron types and fasciculation of the ventral nerve cord are defective. Together, our studies define a new role for HDA-1 in nervous system development, and provide the first evidence for HDAC function in regulating neuronal axon guidance

Developmental decline in neuronal regeneration by the progressive change of two intrinsic timers

Like mammalian neurons, Caenorhabditis elegans neurons lose axon regeneration ability as they age, but it is not known why. Here, we report that let-7 contributes to a developmental decline in anterior ventral microtubule (AVM) axon regeneration. In older AVM axons, let-7 inhibits regeneration by down-regulating LIN-41, an important AVM axon regeneration-promoting factor. Whereas let-7 inhibits lin-41 expression in older neurons through the lin-41 3\u27 untranslated region, lin-41 inhibits let-7 expression in younger neurons through Argonaute ALG-1. This reciprocal inhibition ensures that axon regeneration is inhibited only in older neurons. These findings show that a let-7-lin-41 regulatory circuit, which was previously shown to control timing of events in mitotic stem cell lineages, is reutilized in postmitotic neurons to control postdifferentiation events

Mutations in <i>alg-1</i> suppress precocious development of <i>lin-28(lf)</i> (<i>lin-28(n947)</i>) animals.

<p>(<b>A</b>, <b>B</b>) <i>alg-1</i> mutations suppress the egg-laying defect of <i>lin-28(lf)</i> animals by suppressing the precocious divisions of the vulval precursor cells; (<b>B</b>) Early third larval (eL3) stage animals. Arrowheads indicate vulval cell nuclei. Three vulval precursor cells (P5.p, P6.p, and P7.p) are undivided in the top (N2) and bottom (<i>lin-28(n947); alg-1(ma192)</i>) panels, but in the middle panel (<i>lin-28(n947</i>), P6.p and P7.p have divided twice (one P7.p granddaughter is out of the plane of focus). (<b>C</b>, <b>D</b>) <i>alg-1</i> mutations suppress the <i>lin-28(lf)</i> precocious expression of the adult cell fate marker <i>col-19::gfp</i>. lL4-late fourth larval stage. (<b>E</b>) <i>alg-1</i> mutations also increase the seam cell number (#) produced <i>lin-28(lf)</i> mutant animals (***p<0.0001), and (<b>F</b>) suppress precocious alae formation of L4 animals; dotted line represents absence of the alae, solid line underlines the alae structure. All strains <i>col-19::gfp</i> transgene in the background. n = number of animals scored.</p

Newly isolated <i>alg-1</i> alleles are antimorphic, exhibit retarded development, and display phenotypes more severe than those of <i>alg-1(0)</i>.

<p>(<b>A</b>, <b>B</b>) Bar graphs showing the percent of young adult (YA) animals with wild type alae formation (<b>A</b>) and <i>col-19::gfp</i> adult marker expression (<b>B</b>), where <i>alg-1(anti)</i> alleles have a dosage dependent effect on both phenotypes. (<b>C</b>) Schematic of representative V1–V4 and V6 lineage cell divisions in the wild type, <i>alg-1(anti)</i>, and other heterochronic mutants. (<b>D</b>) <i>alg-1(anti)</i> mutations display increased numbers of seam cells as young adults. ***p<0.001. All strains carry <i>lin-31(lf)</i> and <i>col-19::gfp</i> in the background. The <i>lin-31</i> mutation is present in order to suppress <i>alg-1(anti)</i> vulval bursting phenotypes by non-heterochronic methods. n = number of animals scored.</p

Purified recombinant ALG-1(anti) retain the slicing ability similar to the wild type ALG-1.

<p>(<b>A</b>) SDS-PAGE gel of recombinant ALG-1 proteins. Wild type ALG-1, ALG-1 G553R and ALG-1 S895F were bacterially expressed and purified. (<b>B</b>) Radiolabeled target RNA is cleaved by rALG-1 proteins preloaded with a siRNA (red) to produce a major 15 nt cleavage species as well as two minor cleavage species. (<b>C</b>) rALG-1 proteins, including rALG-1(anti), bind and cleave a perfectly base-paired duplex. (<b>D</b>) rALG-1 proteins bind a microRNA duplex containing containing two mismatches. Pre-bound rALG-1/duplex complexes are able to cleave an RNA target, producing a 15-nt major cleavage RNA species.</p

Four of five newly isolated <i>alg-1</i> alleles carry missense mutations.

<p>(<b>A</b>) A schematic showing the positions of the newly identified <i>alg-1</i> mutations within the ALG-1 protein (black tics) and the existing deletion alleles (red lines). Positions of catalytic sites are indicated by yellow circles. (<b>B</b>) Exon/intron schematic of the two <i>alg-1</i> isoforms predicted and supported by cDNA evidence (Wormbase.org). Boxes represent exonic regions. (<b>C</b>). Western blot analysis on total protein lysate from wild type and <i>alg-1</i> mutant animals. All non-null <i>alg-1</i> alleles, like the wild type, produce 2 isoforms of ALG-1. Newly identified missense alleles of <i>alg-1</i> are marked in red and null alleles are in blue. <i>alg-1(tm369)</i> is a loss of function allele that deletes most of the PIWI domain and produces 2 truncated isoforms of ALG-1(*). All strains with the exception of <i>alg-1(tm492)</i> and <i>alg-1(tm369)</i> carry <i>lin-31(lf)</i> and <i>col-19::gfp</i> in the background.</p

Western blot analysis of ALG-1 immunoprecipitated complexes from extracts of <i>alg-1(anti)</i> and wild type animals.

<p>Immunoprecipitated ALG-1(anti) shows an increased association with DCR-1, and a decreased association with AIN-1, compared to wild type ALG-1. The ratio of DCR-1 to ALG-1 and AIN-1 to ALG-1 were determined by quantitation of the Western blot signals, and each of those ratios for the <i>alg-1(anti)</i> mutants is normalized to that of the wild type. * means not applicable (n/a).</p