Edgar LG et al. (1991) International C. elegans Meeting "'NOBS' AND AN EMBRYONIC ROLE FOR PAL- 1"

Wood WB, Notka F, Wolf N, Edgar LG

[International C. elegans Meeting, 1991]

The embryonic lethal phenotypes ('no back end') resulting from psoralen-induced non-maternal mutations at two loci suggest roles for these genes in posterior embryonic morphogenesis. The 'nob-l' locus maps between spe-6 and unc-25 on the right arm of LGIII. The ct223 allele is a neonatal lethal with a spherical gut and a round posterior, but a normal head; its earliest visible defect is a misarrangement of the E cells in the 100-cell embryo. ct230 is a weaker homozygous viable allele, (approximately 65% fertility) which results in a lumpy deformed tail very similar to vab-7. ct230/eDf2 is nonviable, suggesting that ct223 is a hypomorph, while ct223/eDf2 has the same phenotypic range as ct223/ct223. We have also isolated a larger deficiency of the locus with a break point between spe-6 and 'nob-l' extending at least to bli-S. We are mapping Tcl polymorphisms in this region as an approach to cloning the gene. ct224, which results in a similar but slightly more severe posterior embryonic defect, was isolated in a LGIII screen for more alleles of 'nob-l'. However, it complements 'nob' alleles, maps between dpy17 and unc-79 and fails to complement pal-l, the homeobox gene identified in Cynthia Kenyon's lab, which is involved in postembryonic cell interactions of V6 and T in the male tail. Because ct224 is psoralen-induced and might be a deletion of more than one gene, we are trying to rescue the mutant phenotype by injecting the pal-l plasmids, given us by Cynthia ( thanks!). So far we have no established lines, but we have seen transient rescue in several injection series, including those using the smallest plasmid pWPK6 (5.5 kb). At present we believe that ct224 is a strong, probably a null, allele of pal1, indicating an embryonic role for this gene. We are beginning mosaic analysis to determine which embryonic lineages require the gene function. The double mutant ct223ct224 has a somewhat additive phenotype (hatched embryos look like pears). Construction of double mutants with the weaker phenotypes is planned, and may be more informative as to gene interactions. It is of interest that the homeobox of pal-l shows sequence similarity to that of the caudal gene in Drosophila and its apparent mouse homolog, which also are implicated in posterior/endodermal development.

Pal, Swati et al. (2015) International Worm Meeting "C. elegans CCM-3: A mother's gift to her fledgling that keeps the traffic moving."

PAL, SWATI, Derry, Brent

[International Worm Meeting, 2015]

Cerebral Cavernous malformations (CCMs) are vascular disorders of central nervous system that arise from loss of integrity of blood capillaries causing blood leakage. This leads to variety of symptoms, including headaches, seizures and hemorrhagic stroke. 3 genes causing familial CCM in humans have been identified: CCM1/2/3; CCM3 mutations being most aggressive. Yet the in vivo function of CCM3 is poorly understood. Caenorhabditis elegans contains orthologues of CCM1 (kri-1) and CCM3 (ccm-3). CCM-3 protein is localized along apical membrane of biological tubes (like excretory canal, intestine and germline) and promotes endocytic recycling to maintain membrane integrity (Lant et al., 2015). To investigate the function of CCM-3 in multicellular tube development we have used C. elegans germline as a model.The nematode germ cells undergo incomplete cytokinesis to create openings into a common lumen called the rachis. Proliferation of mitotic stem cells requires GLP-1/Notch. In ccm-3 mutants there are fewer mitotically proliferating cells with greatly reduced GLP-1 levels. RNAi knockdown of sel-9, which regulates recycling of Notch back to the cell surface, rescues the proliferation defects. Ras/MAPK signalling is required for germ cell pachytene exit and rachis formation. Ablation of ccm-3 causes both a reduction in MAPK signalling and collapse of rachis producing undeveloped oocytes. ccm-3 mutants also fail to present vitellogenin receptor RME-2 on oocytes and show defective VIT-2 uptake. Collectively, these results suggest that loss of ccm-3 results in failure of multiple receptors to get trafficked to membrane surfaces, possibly through defects in endocytic recycling. Consistently, we found that CCM-3 colocalizes with the endosome recycling protein RAB-11. Previous work has shown that loss of anillin proteins results in germline defects that are strikingly similar to ccm-3 mutants, suggesting potential interactions between these scaffolding proteins in promoting rachis development. We observed that CCM-3, along with GCK-1, is required for cortical localization of ANI-1 and non-muscle myosin NMY-2. Knockdown of ccm-3 also results in defective mitotic division of the polarized embryo. Based on these results we hypothesize that CCM-3 functions to couple vesicle trafficking and acto-myosin organization to fine-tune proper assembly and function of the germline. Understanding the CCM-3 interactome may help provide general insights into biological tube development and identify non-surgical approaches for treating CCM patients.

PAL, SWATI et al. (2013) International Worm Meeting "Deletion of ccm-3 in C. elegans promotes increased accumulation of reactive oxygen species resulting in apoptosis of germline cells."

Derry, W. Brent, Yu, Bin, PAL, SWATI

[International Worm Meeting, 2013]

Cerebral Cavernous malformations (CCMs) are the disorders of capillaries in the central nervous system that result from a loss of integrity in endothelial cells. CCMs affect approximately 1 in 500 individuals and presents in patients a variety of pathophysiological symptoms that range from mild headaches to severe hemorrhagic stroke. Till date, mutations in three genes have been identified that account for approximately 90% of patients with familial disease, ccm-1, ccm-2 and ccm-3. The most severe prognosis is associated with ccm-3 mutations, but the CCM3 signalling pathway has not been resolved. In contrast to the familial form, sporadic CCMs have not been linked with any genetic loci. The varying degrees of symptoms and severity of disease in patients with the same mutation in either of the familial CCM genes suggests the existence of modifier genes. We have shown that ccm-3 (or C14A4.11) affects multiple tissues in C. elegans. For example, ccm-3 homozygote mutants have truncated excretory canals as well as defective oocyte and spermatocyte development that renders them sterile. The oocytes do not grow to the proper size and undergo massive waves of apoptosis, which can be rescued by expression of wild-type ccm-3 gene. Because mammalian CCM genes have been shown to affect reactive oxygen species (ROS), we cultivated ccm-3 mutants in presence of N-acetyl cysteine (NAC), a ROS scavenger, and observed partial rescue of the germline phenotype. NAC also partially protected germlines of wild-type animals from radiation-induced apoptosis. Because the Ras/MAPK pathway has been shown to promote apoptosis in the germline, we are now examining the status of activated MPK-1/ERK in ccm-3 mutant germlines. CCM-3 physically interacts with the germinal center kinase III (GCK-III) family of proteins to negatively regulate ERK activation and apoptosis in mammals. Ablation of the sole GCK-III gene in the worm, gck-1, also results in defective oocyte development that resembles ccm-3 mutants. We hypothesize that CCM-3 and its binding partner GCK-1 are required for proper growth and survival of oocytes.

Gang, Spencer et al. (2021) International Worm Meeting "Analysis of PALS-25 as an activator of the Intracellular Pathogen Response in C. elegans"

Barkoulas, Michalis, Gang, Spencer, Grover, Manish, Troemel, Emily, Reddy, Kirthi

[International Worm Meeting, 2021]

Tight regulation of immune responses is important for overall fitness, but the mechanisms underlying many of these pathways are incompletely characterized. One example is the recently described "Intracellular Pathogen Response" (IPR) in C. elegans. Our lab has defined the IPR as a set of ~80 genes that are upregulated in response to several triggers including intracellular pathogen infection, proteasome inhibition and heat stress. The IPR is also genetically regulated by the species-specific antagonistic paralogs pals-22 and pals-25. pals-22 represses the IPR; loss-of-function mutations in pals-22 promote IPR gene expression, increase pathogen resistance and improve tolerance of proteotoxic stress. pals-25 acts downstream of pals-22 to activate the IPR; loss-of-function mutations in pals-25 suppress the phenotypes of pals-22 mutants. Recently, we identified a gain-of-function mutation of pals-25 that truncates the C-terminus of PALS-25 by 13 amino acids, or ~5% of the total protein. Unlike previously characterized mutations of pals-25, this pals-25(gf) allele results in constitutive expression of IPR genes in both wild type and pals-22 mutant backgrounds. However, only a subset of IPR genes appear to be upregulated in pals-25(gf) mutants when compared to pals-22 mutants. pals-25(gf) animals are similar to pals-22 mutants in that they are resistant to infection by Nematocida parisii but are dissimilar in that they display wild type tolerance of heat stress. Together, these observations may allow for the identification of IPR-related genes that are specifically important for pathogen resistance phenotypes. Previous co-IP/MS studies determined that PALS-22 and PALS-25 are physically associated. Here we show that FLAG-IP of PALS-22::GFP::3xFLAG identifies PALS-25 as a binding partner, but this interaction is no longer detected for the C-terminally truncated version of PALS-25 encoded by the pals-25(gf) allele. Yeast two-hybrid analysis of full-length PALS-25 also revealed PALS-22 as a binding partner, but the truncated version of PALS-25 did not identify PALS-22 as a binding partner. Together, our results suggest a model where PALS-22 physically represses the ability of PALS-25 to activate the IPR and the interaction of the two proteins requires the C-terminus of PALS-25. Ongoing studies will explore the mechanism of IPR activation by PALS-25 after it is released from repression by PALS-22.

Edgar LG et al. (1996) West Coast Worm Meeting "EMBRYONIC FUNCTIONS OF pal-1"

Edgar LG, Wood WB

[West Coast Worm Meeting, 1996]

The worm caudal homolog pal-1 functions maternally to determine posterior fates in the C and D lineages (C. Hunter and C. Kenyon, personal communication). The non-maternal small deletion mutations ct224 and ct281 (presumed null mutations) result in an L1 Nob phenotype with severe posterior defects (Edgar et al, C. elegans Meeting Abstracts, 1993) and some less severe anterior malformations. We are currently investigating the embryonic expression of pal-1 and the defects causing the Nob phenotype by lineage and marker analysis of the mutants. By antibody staining (anti-PAL-1 antibodies a much-appreciated gift from Craig Hunter) and by expression of pal-1::lacZ constructs, the pal-1 gene is embryonically expressed from about the 28-cell stage, increasing as maternal protein disappears, in a number of posterior cells derived from AB, C, and E lineages, and in only 4 anterior cells, Z1, Z4, mu intR, and M, which migrate to the posterior shortly before morphogenesis. During morphogenesis, ~40-60 cells in the ventral posterior region stain with antibody. By 4D lineage analysis of mutant embryos (n=3), all cells appeared to be born, with one exception late in the C lineage. However, abnormal positions and cell contacts were observed in the posterior hypodermal AB and C lineages as early as the 4C stage. In the C hypodermal lineage, the double row of mid-dorsal cells, normally oriented along the a-p axis, was skewed; the contralateral nuclear migration in the posterior hyp7 cells before their fusion did not occur; and some premature cell fusions took place. The ABarpp-derived dorsal hypodermal pair just anterior to the C hypodermals also showed misalignment and no nuclear migration (characteristic of the more anterior ABarpa-derived hypodermal cells. Staining for a seam cell marker (wIS1, a gift from Joel Rothman) indicates that the number of seam cells is significantly lower in Nobs than in wild-type embryos, strengthening the idea that some V cells undergo a fate transformation from one type of hypodermal cell to another. Our current hypothesis is that mis-specification of hypodermal C cells and AB cells that they contact at the 28-50 cell stages somehow lead to the Nob phenotype, perhaps because of the defects observed in hypodermal cell shape changes. Based on these defects and the PAL-1 expression pattern, embryonic pal-1 function may be required to specify cell identities along both the a-p and d-v axes. The apparent similarities between this function and the functions of caudal and its homologues in other embryos suggest that these genes are involved in an ancient and highly conserved embryonic patterning process.

Hunter CP (1997) International C. elegans Meeting "Targeting pal-1 translation to posterior blastomeres"

Hunter CP

[International C. elegans Meeting, 1997]

The C. elegans pal-1 gene encodes a caudal homolog that specifies posterior blastomere identity. pal-1 activity is targeted to specific blastomeres by multiple regulatory steps. The first step is to repress translation of maternal pal-1 mRNA in oocytes and early embryos. Then, at the 4-cell stage, pal-1 translation is specifically derepressed in the two posterior blastomeres. The repression and derepression of pal-1 translation is controlled, at least in part, by the pal-1 3'UTR. Injected lacZ RNA that includes the pal-1 3' UTR is preferentially expressed in posterior blastomeres. We have identified several genes that are required to repress and derepress pal-1 translation. mex-3 encodes a putative RNA binding protein that is required to repress pal-1 translation. In mex-3 mutant embryos PAL-1 protein is detected in oocytes and all early blastomeres. In contrast par-1 and par-4 functions are required for derepression of the mex-3 mediated repression. In par-1 or par-4 single mutant embryos PAL-1 is not detected, but in mex-3; par-1 and mex-3; par-4 double mutant embryos PAL-1 is detected in all blastomeres. Finally, par-3 activity appears to be required to localize the derepression activity to the posterior blastomeres. In par-3 mutant embryos pal-1 translation appears to be properly repressed until the 4-cell stage. However, in 4-cell and older par-3 mutant embryos PAL-1 can be detected in all blastomeres. We are presently screening for additional mutations that alter the PAL-1 expression pattern and are analyzing the translational control regions in the pal-1 mRNA.

Edgar LG et al. (1997) International C. elegans Meeting "EMBRYONIC FUNCTIONS OF PAL-1"

Gonzales R, Wood WB, Edgar LG, Wang H

[International C. elegans Meeting, 1997]

The caudal homolog pal-1 is expressed both maternally and embryonically. Maternally it functions to specify muscle fate in the C and D lineages (1). Embryonic loss of function results in the severely posteriorly malformed Nob phenotype (2). We are using reporter constructs, 4D lineage analysis, and mosaic analysis of the null allele ct224 to continue characterization of pal-1 embryonic function. Lineage analysis shows defects in the C hypodermal lineages (which normally express pal-1): cells are misplaced from the 4-C-cell stage onward, and the normal contralateral nuclear migration does not occur. Adjacent AB posterior hypodermal cells (which normally do not express pal-1 at these stages), are also mispositioned, and expression of the seam cell-specific reporter wIS1 is reduced in the posterior. Although C and D muscle lineages appear normal, preliminary results indicate that the number of cells expressing a myo-3::GFP reporter is reduced, suggesting a function for pal-1 in the C muscle lineage, even though wild-type embryonic expression of pal-1 is detectable in this lineage only briefly if at all with antibodies and a GFP construct. This hypothesis is supported by preliminary mosaic analysis: losses in C muscle lineages cause an intermediate lumpy phenotype localized in the area of loss. Other functional foci appear to lie in ABp (pal-1-expressing progeny contribute to the rectal tissues) and the C hypodermal lineage. Thus there may be more than one way to make a Nob. Based on the finding that a transgenic construct containing pal-1 exons 1 and 2 (excluding the homeodomain) causes a dominant-negative Nob phenotype, we are beginning a yeast two-hybrid screen, using Pal-1 as bait, to identify possible interacting proteins, one of which could be MyoD (3). Pal-1 does not appear to interact with itself. Initially positive clones from the Okkema embryonic library are currently being tested further. (1) C. Hunter and C. Kenyon, Cell 87: 217-226, 1996. (2) L. Edgar, S. Carr and B. Wood, 1996 International Worm Meeting Abstr. 198. (3) M. Park, R. Littlejohn and M. Krause, WBG 14(5) 1997.

Darcy E Mootz et al. (2002) East Coast Worm Meeting "GLD-1 and the pal-1 3' UTR are implicated in the post-initiation translational repression of maternal pal-1 mRNA in C. elegans"

Darcy E Mootz, Diana Ho, Craig P Hunter

[East Coast Worm Meeting, 2002]

The regulation of mRNA translation is a prominent, yet poorly understood mechanism of gene control. To gain insight into the molecular mechanisms of translational control, we are investigating the translational regulation of maternal pal-1 mRNA, which encodes a conserved homeodomain protein involved in posterior patterning of the C. elegans embryo. We have focused on translational control in the distal gonad arms of adult hermaphrodites (where pal-1 mRNA is present, yet no PAL-1 protein is detected), and have gained insight into 3' UTR regulatory elements, a trans-acting factor, and the level of the translational block. To identify translational control elements in pal-1 mRNA, we used a lacZ reporter RNA assay. We found that the pal-1 3' UTR is sufficient to repress the translation of a lacZ reporter RNA (lacZ::pal-1 3' UTR) in the distal gonad arms of adult hermaphrodites. Through deletion analysis of the lacZ::pal-1 3' UTR RNA, we identified two regions of approximately 180 nucleotides that repress distal germline expression. Subdivision of one of these regions identified a potential translational activation element as well as a 108-nucleotide germline repression element, the GRE, which is both necessary and sufficient for robust germline repression. Through a candidate gene approach, we identified GLD-1, a maxi-KH domain protein, as a negative regulator of PAL-1 expression. In gld-1 null mutants, ectopic PAL-1 immunofluorescence is detected in the distal gonad arms of adult hermaphrodites. In addition, gld-1 can act through the GRE to repress distal germline expression; a gfp transgene under the control of the GRE is ectopically expressed in distal gonad arms when gld-1 activity is reduced through RNAi. Finally, to begin addressing the molecular mechanism of repression, we performed polysome analysis to determine where pal-1 mRNA from the distal germline fractionates with respect to ribosomes. Multiple experiments suggest that pal-1 translation in the distal germline may be blocked after pal-1 mRNA is loaded on ribosomes. Since initiation is often the rate-limiting step in protein synthesis, the loading of pal-1 mRNA on ribosomes in the germline may allow for the rapid accumulation of PAL-1 protein in the 4-cell embryo when the translational block is relieved.

DE Mootz et al. (1999) International C. elegans Meeting "Identification of cis- and trans- acting factors controlling the translation of maternal pal-1 mRNA"

CH Hunter, DE Mootz

[International C. elegans Meeting, 1999]

Proper patterning of the C.elegans embryo relies on the posterior localization of PAL-1. Two observations suggest this posterior localization is due to the regulated translation of maternal pal-1 mRNA. First, pal-1 mRNA is present in oocytes and all cells of the early embryo, whereas PAL-1 protein is not detected until the 4-cell stage. Second, the pal-1 3' UTR can restrict the translation of a reporter RNA ( lacZ::pal-1 3' UTR) to posterior blastomeres. We are interested in characterizing the molecular mechanisms underlying the spatial and temporal control of pal-1 translation. Towards this end, we are trying to identify the translational control elements in the pal-1 message, as well as the factors that bind these elements. To identify control elements, we are performing deletion analysis of a lacZ reporter RNA bearing the pal-1 5' and 3' UTRs. To complement this analysis, we are looking for regions of pal-1 5' and 3' UTRs that are evolutionarily conserved. Thus far, we have cloned the pal-1 3' UTR in C.remanei and C.briggsae , and have found a 36 nucleotide element that is highly conserved between all three species. We are testing the functional significance of this element using the lacZ reporter RNA assay, and we intend to use this element as bait in genetic and biochemical schemes to identify proteins that specifically interact with pal-1 mRNA. To date, our efforts to identify translational regulators that bind pal-1 mRNA have focused on MEX-3. mex-3 encodes a putative RNA binding protein containing two KH domains (Draper et al., 1996), and genetic studies have suggested that it functions through the pal-1 3' UTR to repress pal-1 translation. In order to determine whether MEX-3 is a direct regulator of pal-1 translation, we are testing whether MEX-3 specifically associates with pal-1 mRNA, either alone, or in a complex with other proteins. We have shown that recombinant MEX-3 is capable of binding the pal-1 3'UTR in vitro , and we are testing the specificity of this interaction. In addition, we are performing both in vitro and in vivo experiments to test for the presence of MEX-3 in a pal-1 mRNA binding complex. Draper, B.W., C.C.Mello, B.Bowerman, J.Hardin, and J.R.Priess (1996). Cell 87: 205-216.

Michael C Fitzgerald et al. (2001) International C. elegans Meeting "Role of MEX-3 in Patterning PAL-1 Expression"

Michael C Fitzgerald, Craig P Hunter

[International C. elegans Meeting, 2001]

PAL-1 is a caudal-like homeodomain protein that is first expressed at the 4-cell stage in the two posterior blastomeres. This spatial and temporal patterning of PAL-1 expression is dependent on the putative RNA binding protein MEX-3 and the pal-1 3' untranslated region (UTR). A large body of circumstantial evidence suggests that MEX-3 may repress pal-1 translation by binding to the pal-1 3' UTR. We are taking a biochemical approach to test this possibility directly. Current efforts focus on using reversible RNA-protein cross-linking, followed by immunoprecipitation of MEX-3, and RT-PCR analysis of the co-precipitated RNA. Positive results in this assay will be followed by experiments to investigate the mechanisms by which MEX-3 patterns PAL-1 expression.