Wnt signaling chapter in WormBook
Among those papers is the reference for the reporter gene fusion strain (JRW35) that we used (Kalis2014_VNC_patterning.pdf) and the gene used in the reporter fusion transgenic: tph-1 (Sze2000.pdf). There is also an article about the C. elegans HOX complex (Aboobaker2003.pdf), which, of course includes the lin-39 gene. I do not have full pdfs for some of the older papers found below, so an abstract is provided here. You may be able to find some of these if needed via PubMed or Google Scholar.
A clear and concise review of lin-39's role in vulval development can be found in the introduction of Gradien & Sommer, 2001 (Gradien2001.pdf), an evo-devo paper comparing the functions of lin-39 in C.e. and a different species of nematode.
Hox genes control the choice of cell fates along the anteroposterior (AP) body axis of many organisms. In C. elegans, two Hox genes, lin-39 and mab-5, control the cell fusion decision of the 12 ventrally located Pn.p cells. Specific Pn.p cells fuse with an epidermal syncytium, hyp7, in a sexually dimorphic pattern. In hermaphrodites, Pn.p cells in the mid-body region remain unfused whereas in males, Pn.p cells adopt an alternating pattern of syncytial and unfused fates. The complexity of these fusion patterns arises because the activities of these two Hox proteins are regulated in a sex-specific manner. MAB-5 activity is inhibited in hermaphrodite Pn.p cells and thus MAB-5 normally only affects the male Pn.p fusion pattern. Here we identify a gene, ref-1, that regulates the hermaphrodite Pn.p cell fusion pattern largely by regulating MAB-5 activity in these cells. Mutation of ref-1 also affects the fate of other epidermal cells in distinct AP body regions. ref-1 encodes a protein with two basic helix-loop-helix domains distantly related to those of the hairy/Enhancer of split family. ref-1, and another hairy homolog, lin-22, regulate similar cell fate decisions in different body regions along the C. elegans AP body axis.
During larval development in C. elegans, some of the cells of the ventral epidermis, the Pn.p cells, fuse with the growing epidermal syncytium hyp7. The pattern of these cell fusions is regulated in a complex, sexually dimorphic manner. It is essential that some Pn.p cells remain unfused in order for some sex-specific mating structures to be generated. The pattern of Pn.p cell fusion is regulated combinatorially by two genes of the C. elegans Hox gene cluster: lin-39 and mab-5. Some of the complexity in the Pn.p cell fusion pattern arises because these two Hox proteins can regulate each other's activities. We describe a zinc-finger transcription factor, REF-2, that is required for the Pn.p cells to be generated and to remain unfused. REF-2 functions with the Hox proteins to prevent Pn.p cell fusion. ref-2 may also be a transcriptional target of the Hox proteins.
Abstract: Cells in the mid-body region of the nematode C. elegans develop differently from their anterior or posterior homologs. The gene lin-39 is required for mid-body region-specific development. In lin-39 mutants, mid-body cells express fates characteristic of more anterior or posterior homologs, and the migration of a neuroblast through the mid-body is defective. lin-39 acts cell autonomously in these mid-body cells and in the migrating neuroblast. lin-39 encodes a protein with an Antennapedia class homeodomain, most similar to those of the Drosophila homeotic genes Deformed and Sex combs reduced, and is located in a homeotic gene cluster with two other regional homeotic genes, mab-5 and egl-5. lin-39 and mab-5 function combinatorially in 2 ectodermal cells and have redundant functions in gonad development.
Hox genes pattern the fates of the ventral ectodermal Pn.p cells that lie along the anteroposterior (A/P) body axis of C. elegans. In these cells, the Hox genes are expressed in sequential overlapping domains where they control the ability of each Pn.p cell to fuse with the surrounding syncytial epidermis. The activities of Hox proteins are sex-specific in this tissue, resulting in sex-specific patterns of cell fusion: in hermaphrodites, the mid-body cells remain unfused, whereas in males, alternating domains of syncytial and unfused cells develop. We have found that the gene egl-27, which encodes a C. elegans homologue of a chromatin regulatory factor, specifies these patterns by regulating both Hox gene expression and Hox protein function. In egl-27 mutants, the expression domains of Hox genes in these cells are shifted posteriorly, suggesting that egl-27 influences A/P positional information. In addition, egl-27 controls Hox protein function in the Pn.p cells in two ways: in hermaphrodites it inhibits MAB-5 activity, whereas in males it permits a combinatorial interaction between LIN-39 and MAB-5. Thus, by selectively modifying the activities of Hox proteins, egl-27 elaborates a simple Hox expression pattern into complex patterns of cell fates. Taken together, these results implicate egl-27 in the diversification of cell fates along the A/P axis and suggest that chromatin reorganization is necessary for controlling Hox gene expression and Hox protein function.
In C. elegans, the epithelial Pn.p cells adopt either a vulval precursor cell fate or fuse with the surrounding hypodermis (the F fate). Our results suggest that a Wnt signal transduced through a pathway involving the beta-catenin homolog BAR-1 controls whether P3.p through P8.p adopt the vulval precursor cell fate. In bar-1 mutants, P3.p through P8.p can adopt F fates instead of vulval precursor cell fates. The Wnt/bar-1 signaling pathway acts by regulating the expression of the Hox gene lin-39, since bar-1 is required for LIN-39 expression and forced lin-39 expression rescues the bar-1 mutant phenotype. LIN-39 activity is also regulated by the anchor cell signal/let-23 receptor tyrosine kinase/let-60 Ras signaling pathway. Our genetic and molecular experiments show that the vulval precursor cells can integrate the input from the BAR-1 and LET-60 Ras signaling pathways by coordinately regulating activity of the common target LIN-39 Hox.
The fate of ventral epidermal cells differs among nematode species. Nonvulval cells fuse with the epidermis in Caenorhabditis elegans, whereas the homologous cells undergo apoptosis in Pristionchus pacificus. The homeotic gene lin-39 is involved in the regulation of these epidermal cell fates. In Caenorhabditis, lin-39 prevents cell fusion of potential vulval cells and specifies the vulva equivalence group. Pristionchus vulvaless mutants that displayed apoptosis of the vulval precursor cells were isolated, and point mutations in lin-39 were identified. Thus, the evolution of these epidermal cell fates is driven by different intrinsic properties of homologous cells.
Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.
During Caenorhabditis elegans vulval development, activation of receptor tyrosine kinase/Ras and Notch signaling pathways causes three vulval precursor cells (VPCs) to adopt induced cell fates. A Wnt signaling pathway also acts in cell fate specification by the VPCs, via regulation of the Hox gene lin-39. We show here that either mutation of pry-1 or expression of an activated BAR-1 -catenin protein causes an Overinduced phenotype, in which greater than three VPCs adopt induced cell fates. This indicates that pry-1, which encodes a C. elegans axin homolog, acts as a negative regulator of Wnt signaling in the VPCs. Loss of activity of the APC homolog apr-1 increases the penetrance of this Overinduced phenotype, suggesting that APR-1 may play a negative role in Wnt signaling in this process in C. elegans similar to APC proteins in other systems. The Overinduced phenotype is suppressed by reduction of function of the genes pop-1 TCF and lin-39 Hox. Surprisingly, the Overinduced phenotype caused by hyperactivated Wnt signaling is not dependent on signaling through the Ras pathway. These data suggest that hyperactivation of Wnt signaling is sufficient to cause VPCs to adopt induced fates and that a canonical Wnt pathway may play an important role during C. elegans vulval induction.
Vulval cell-fate determination in Caenorhabditis elegans requires the action of numerous gene products, including components of the Ras/Raf/MAPK signaling cascade and the hox gene lin-39. sem-4 encodes a zinc finger protein with previously characterized roles in fate specification of sex myoblasts, coelomocytes, and multiple neuronal lineages in C. elegans (M. Basson and R. Horvitz, 1996, Genes Dev. 10, 1953-1965). By characterizing three new alleles of sem-4 that we identified in a screen for vulval-defective mutants, we determined that loss of sem-4 activity results in abnormal specification of the secondary vulval cell lineages. We analyzed sem-4 interactions with other genes involved in vulval differentiation and determined that sem-4 does not function directly in the Ras-mediated signal transduction pathway but acts in close association with and upstream of lin-39 to promote vulval cell fate. We demonstrate that sem-4 regulates lin-39 expression and propose that sem-4 is a regulator of lin-39 in the vulval cell-fate determination pathway that may act to link lin-39 to incoming signals.
Inactivation of the Caenorhabditis elegans APC-related gene (apr-1) has pointed at two separate functions of apr-1. First, apr-1 is required for the migration of epithelial cells during morphogenesis of the embryo. In this process, APR-1 may act in a Cadherin/alpha-Catenin/beta-Catenin complex as a component of adherens junctions. Second, apr-1 is required for Hox gene expression, most likely by positively regulating the activity of the Wingless signaling pathway. During embryogenesis, apr-1 is required for the expression of ceh-13 labial in anterior seam and muscle cells and during larval development, apr-1 is necessary for the expression of lin-39 deformed in the vulval precursor cells. Thus, APR-1 may positively regulate the activity of the beta-Catenin/Armadillo-related proteins HMP-2 in migrating epithelial cells and BAR-1 in the vulval precursor cells.
Antennapedia class homeobox (Hox) genes specify cell fates in successive anteroposterior body domains in vertebrates, insects and nematodes1-3. The DNA-binding homeodomain sequences are very similar between vertebrate and Drosophila Hox proteins, and this similarity allows vertebrate Hox proteins to function in Drosophila4-7. In contrast, the Caenorhabditis elegans homeodomains are substantially divergent8. Further, C. elegans differs from both insects and vertebrates in having a non-segmented body as well as a distinctive mode of development that involves asymmetric early cleavages and invariant cell lineages. Here we report that, despite these differences, Drosophila Hox proteins expressed in C. elegans can substitute for C. elegans Hox proteins in the control of three different cell-fate decisions: the regulation of cell migra-tion, the specification of serotonergic neurons, and the specification of a sensory structure. We also show that the specificity of one C elegans Hox protein is partly determined by two amino acids that have been implicated in sequence-specific DNA binding. Together these findings suggest that factors important for target recognition by specific Hox proteins have been conserved throughout much of the animal kingdom.
Background: Reproduction in animals requires development of distinct neurons in each sex. In C. elegans,
most ventral cord neurons (VCNs) are present in both sexes, with the exception of six hermaphrodite-specific
neurons (VCs) and nine pairs of male-specific neurons (CAs and CPs) that arise from analogous precursor cells.
How are the activities of sexual regulators and mediators of neuronal survival, division, and fate coordinated
to generate sex-specificity in VCNs? Results: To address this, we have developed a toolkit of VCN markers that
allows us to examine sex-specific neurogenesis, asymmetric fates of daughters of a neuroblast division, and
regional specification on the anteroposterior axis. Here, we describe the roles of the Hox transcription factors
LIN-39 and MAB-5 in promoting survival, differentiation, and regionalization of VCNs. We also find that the
TALE class homeodomain proteins CEH-20 and UNC-62 contribute to specification of neurotransmitter fate in
males. Furthermore, we identify that VCN sex is determined during the L1 larval stage. Conclusions: These
findings, combined with future analyses made possible by the suite of VCN markers described here, will elucidate
how Hox-mediated cell fate decisions and sex determination intersect to influence development of neuronal
sex differences.
A principal challenge currently facing biologists is how to connect the complete DNA sequence of an organism to its development and behaviour.
Large-scale targeted-deletions have been successful in defining gene functions in the single-celled yeast Saccharomyces cerevisiae, but
comparable analyses have yet to be performed in an animal. Here we describe the use of RNA interference to inhibit the function of ~86% of the
19,427 predicted genes of C. elegans. We identified mutant phenotypes for 1,722 genes, about two-thirds of which were not previously associated
with a phenotype. We find that genes of similar functions are clustered in distinct, multi-megabase regions of individual chromosomes; genes in
these regions tend to share transcriptional profiles. Our resulting data set and reusable RNAi library of 16,757 bacterial clones will facilitate
systematic analyses of the connections among gene sequence, chromosomal location and gene function in C. elegans.
Development of the vulva in C. elegans is mediated by the combinatorial action of several convergent regulatory inputs, three of which, the Ras, Wnt and Rb-related pathways, act by regulating expression of the lin-39 Hox gene. LIN-39 specifies cell fates and regulates cell fusion in the mid-body region, leading to formation of the vulva. In the lateral seam epidermis, differentiation and cell fusion have been shown to be regulated by two GATA-type transcription factors, ELT-5 and -6. We report that ELT-5 is encoded by the egl-18 gene, which was previously shown to promote formation of a functional vulva. Furthermore, we find that EGL-18 (ELT-5), and its paralogue ELT-6, are redundantly required to regulate cell fates and fusion in the vulval primordium and are essential to form a vulva. Elimination of egl-18 and elt-6 activity results in arrest by the first larval stage; however, in animals rescued for this larval lethality by expression of ELT-6 in non-vulval cells, the post-embryonic cells (P3.p-P8.p) that normally become vulval precursor cells often fuse with the surrounding epidermal syncytium or undergo fewer than normal cell divisions, reminiscent of lin-39 mutants. Moreover, egl-18/elt-6 reporter gene expression in the developing vulva is attenuated in lin-39(rf) mutants, and overexpression of egl-18 can partially rescue the vulval defects caused by reduced lin-39 activity. LIN-39/CEH-20 heterodimers bind two consensus HOX/PBC sites in a vulval enhancer region of egl-18/elt-6, one of which is essential for vulval expression of egl-18/elt-6 reporter constructs. These findings demonstrate that the EGL-18 and ELT-6 GATA factors are essential, genetically redundant regulators of cell fates and fusion in the developing vulva and are apparent direct transcriptional targets of the LIN-39 Hox protein.
The fan and rays of the C. elegans male tail constitute a compound sensory organ essential for mating. Within this organ, the individual sensilla, known as rays, have unique identities. We show that ray identities are patterned by a selector gene mechanism in a manner similar to other serially homologous axial structures. One selector gene that promotes the identities of a subset of the rays is the Hox gene egl-5. Within EGL-5-expressing rays, further patterning is provided by a Pax-6 homolog and a signal of the TGFbeta family. These genes and pathway coordinately specify multiple ray properties affecting all three terminal ray cell types. These properties include complex patterns of FMRFamide-like (FaRP) neuropeptides, serotonin (5HT) and dopamine expression, and ray morphology. Differences in these differentiated characteristics give each sensillum a unique identity and potentially endow the compound ray organ with a higher-order information gathering capacity.
Defining a behavior that requires the function of specific neurons in the free-living nematode C. elegans can allow one to screen for mutations that disrupt the specification or function of those neurons. We identified serotonin-immunoreactive neurons required for tail curling or "turning" behavior exhibited by C. elegans males during mating. Males mutant in three different genes that reduce serotonin expression, cat-1, cat-4, and bas-1, exhibited defects in turning behavior similar to those of wild-type males in which these neurons were ablated. The turning defect of cat-4 males was rescued by exogenous serotonin, consistent with the idea that their behavioral defect is caused by a lack of serotonin. While the serotonin-deficient mutants we analyzed shared certain behavioral traits, they were blocked for serotonin synthesis or handling at different steps. Analysis of these and additional serotonin-deficient mutants may help us understand how a neuron controls the expression of a serotonergic phenotype.
The Ras signaling pathway specifies a variety of cell fates in many organisms. However, little is known about the genes that function downstream of the conserved signaling cassette, or what imparts the specificity necessary to cause Ras activation to trigger different responses in different tissues. In C. elegans, activation of the Ras pathway induces cells in the central body region to generate the vulva. Vulval induction takes place in the domain of the Hox gene lin-39. We have found that lin-39 is absolutely required for Ras signaling to induce vulval development. During vulval induction, the Ras pathway, together with basal lin-39 activity, up-regulates lin-39 expression in vulval precursor cells. We find that if lin-39 function is absent at this time, no vulval cell divisions occur. Furthermore, if lin-39 is replaced with the posterior Hox gene mab-5, then posterior structures are induced instead of a vulva. Our findings suggest that in addition to permitting vulval cell divisions to occur, lin-39 is also required to specify the outcome of Ras signaling by selectively activating vulva-specific genes.
Characterizing the functions of the many genes discovered by the sequencing projects is now the primary focus of genome-scale studies. Although sequence or structure-based comparisons are helping to generate hypotheses on the biochemical functions of many gene products, determining the in vivo role(s) for large sets of genes remains a critical objective. RNA interference (RNAi) offers a rapid way to gain a first look at loss-of-function phenotypes associated with specific genes. So far RNAi has been used to test the function of a third of the predicted genes in the Caenorhabditis elegans (C. elegans) genome, and it can be expected that a first pass survey of the entire genome will soon be completed. From the current body of work an initial estimate of the power and challenges of using RNAi for genome-wide analyses can be made. A comparison of results obtained from independent large-scale RNAi studies reveals that despite a high degree of congruence, no single study is likely to achieve a comprehensive RNAi-based phenotypic ÒmapÓ of the C. elegans genome; instead a more accurate picture will be assembled from a composite of independent results for the same genes. RNAi analysis, together with other functional genomic approaches such as expression profiling and protein interaction mapping, is transforming C. elegans into a premier model system for the development and integration of functional genomic approaches in a metazoan.
Intricate patterns of overlapping HOM-C gene expression along the A/P axis have been observed in many organisms; however, the significance of these patterns in establishing the ultimate fates of individual cells is not well understood. We have examined the expression of the Caenorhabditis elegans Antennapedia homolog mab-5 and its role in specifying cell fates in the posterior of the ventral nerve cord. We find that the pattern of fates specified by mab-5 not only depends on mab-5 expression but also on post-translational interactions with the neighboring HOM-C gene lin-39 and a second, inferred gene activity. Where mab-5 expression overlaps with lin-39 activity, they can interact in two different ways depending on the cell type: They can either effectively neutralize one another where they are both expressed or lin- 39 can predominate over mab-5. As observed for Antennapedia in Drosophila, expression of mab-5 itself is repressed by the next most posterior HOM-C gene, egl-5. Thus, a surprising diversity in HOM-C regulatory mechanisms exists within a small set of cells even in a simple organism.
General mechanisms by which Hox genes establish cell fates are known. However, a few Hox effectors mediating cell behaviors have been identified. Here we found the first effector of LIN-39/HoxD4/Dfd in Caenorhabditis elegans. In specific vulval precursor cells (VPCs), LIN-39 represses early and late expression of EFF-1, a membrane protein essential for cell fusion. Repression of eff-1 is also achieved by the activity of CEH-20/Exd/Pbx, a known cofactor of Hox proteins. Unfused VPCs in lin-39(-);eff-1(-) double mutants fail to divide but migrate, executing vulval fates. Thus, lin-39 is essential for inhibition of EFF-1-dependent cell fusion and stimulation of cell proliferation during vulva formation.
short article, no abstract available
Genetic interference mediated by double-stranded RNA (RNAi) has been a valuable tool in the analysis of gene function in Caenorhabditis elegans. Here we report an efficient induction of RNAi using bacteria to deliver double-stranded RNA. This method makes use of bacteria that are deficient in RNaseIII, an enzyme that normally degrades a majority of dsRNAs in the bacterial cell. Bacteria deficient for RNaseIII were engineered to produce high quantities of specific dsRNA segments. When fed to C. elegans, such engineered bacteria were found to produce populations of RNAi-affected animals with phenotypes that were comparable in expressivity to the corresponding loss-of-function mutants. We found the method to be most effective in inducing RNAi for non-neuronal tissue of late larval and adult hermaphrodites, with decreased effectiveness in the nervous system, in early larval stages, and in males. Bacteria-induced RNAi phenotypes could be maintained over the course of several generations with continuous feeding, allowing for convenient assessments of the biological consequences of specific genetic interference and of continuous exposure to dsRNAs
The molecular mechanisms underlying the formation of neurons with defined neurotransmitters are not well understood. In this study, we demonstrate that the PcG-like genes in Caenorhabditis elegans, sop-2 and sor-3, regulate the formation of dopaminergic and serotonergic neurons and several other neuronal properties. sor-3 encodes a novel protein containing an MBT repeat, a domain that contains histone-binding activity and is present in PcG proteins SCM and Sfmbt in other organisms. We further show that mutations in sor-3 lead to ectopic expression of Hox genes and cause homeotic transformations. Specification of certain neuronal identities by these PcG-like genes appears to involve regulation of non-Hox gene targets. Our studies revealed that the PcG-like genes are crucial for coordinately regulating the expression of discrete aspects of neuronal identities in C. elegans.
We describe the properties of a new gene, sop-3, that is required for the regulated expression of a C. elegans Hox gene, egl-5, in a postembryonic neuroectodermal cell lineage. Regulated expression of egl-5 in this cell lineage is necessary for development of the sensory rays of the male tail. sop-3 encodes a predicted novel protein of 1475 amino acids without clear homologs in other organisms. However, the sequence contains motifs consisting of homopolymeric runs of amino acids found in several other transcriptional regulators, some of which also act in Hox gene regulatory pathways. The genetic properties of sop-3 are very similar to those of sop-1, which encodes a component of the transcriptional Mediator complex, and mutations in the two genes are synthetic lethal. This suggests that SOP-3 may act at the level of the Mediator complex in regulating transcription initiation. In a sop-3 loss-of-function background, egl-5 is expressed ectopically in lineage branches that normally do not express this gene. Such expression is dependent on the Hox gene mab-5, as it is in branches where egl-5 is normally expressed. Ectopic egl-5 expression is also dependent on the Wnt pathway. Thus, sop-3 contributes to the combinatorial control of egl-5 by blocking egl-5 activation by MAB-5 and the Wnt pathway in inappropriate lineage branches.
Polycomb group (PcG)-mediated repression of C. elegans Hox genes has not been demonstrated, and genes homologous to components of one of the PcG complexes (PRC1) have not been identified in the C. elegans genome. We find that a mechanism of general Hox gene repression exists in C. elegans, carried out in part by SOP-2, a protein related to, but not orthologous with, any PcG protein. sop-2 mutations lead to widespread ectopic expression of Hox genes and homeotic transformations. SOP-2 contains a SAM domain, a self-associating protein domain found in other repressors, including a core component of PRC1 and ETS transcription factors. Phylogenetic analysis indicates that this domain is more closely related to those of the ETS family than to those of PcG proteins. The results suggest that global repression of Hox genes has been taken over by a different branch of the SAM domain family during the evolution of nematodes.
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The zinc finger protein REF-2 functions with the Hox genes to inhibit cell fusion in the ventral epidermis of C. elegans
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Control of cell fates in the central body region of C. elegans by the homeobox gene lin-39
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The beta-catenin homolog BAR-1 and LET-60 Ras coordinately regulate the Hox gene lin-39 during Caenorhabditis elegans vulval development
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Hox transcription factors have been implicated in playing a central role in the evolution of animal morphology. Many studies indicate the evolutionary importance of regulatory changes in Hox genes, but little is known about the role of functional changes in Hox proteins. In the nematodes Pristionchus pacificus and Caenorhabditis elegans, developmental processes can be compared at the cellular, genetic, and molecular levels and differences in gene function can be identified. The Hox gene lin-39 is involved in the regulation of nematode vulva development. Comparison of known lin-39 mutations in P. pacificus and C. elegans revealed both conservation and changes of gene function. Here, we study evolutionary changes of lin-39 function using hybrid transgenes and site-directed mutagenesis in an in vivo assay using C. elegans lin-39 mutants. Our data show that despite the functional differences of LIN-39 between the two species, Ppa-LIN-39, when driven by Cel-lin-39 regulatory elements, can functionally replace Cel-lin-39. Furthermore, we show that the MAPK docking and phosphorylation motifs unique for Cel-LIN-39 are dispensable for Cel-lin-39 function. Therefore, the evolution of lin-39 function is driven by changes in regulatory elements rather than changes in the protein itself.
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Sem-4 promotes vulval cell-fate determination in Caenorhabditis elegans through regulation of lin-39 Hox
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The novel C. elegans gene sop-3 modulates Wnt signaling to regulate Hox gene expression
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