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.

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.

Gilbert, Sophie P. R. et al. (2013) International Worm Meeting "Defining the role of the Caenorhabditis elegans homeobox protein, PAL-1, in the development of the stem-like seam cells."

Brabin, Charles, Appleford, Peter J., Woollard, Alison, Gilbert, Sophie P. R.

[International Worm Meeting, 2013]

pal-1, the C. elegans homologue of Drosophila caudal, has previously been shown to have an important role in establishing posterior patterning during embryogenesis 1. Here we investigate a novel role for pal-1 during seam cell development. pal-1 was identified in a small scale candidate RNAi screen to detect genes acting redundantly with rnt-1, a gene already known to be essential for maintaining correct seam cell divisions. Synthetic lethality of pal-1 with rnt-1 was observed, indicating that the two gene products may physically interact during embryogenesis. Although pal-1 null animals are embryonic lethal, worms homozygous for a pal-1 mutant allele e2091 survive embryogenesis but on hatching display an uneven distribution of seam cells along their length, together with other seam defects. Analysis of this strain has previously identified two point mutations in the last intron of pal-1 2; we have found that a wild-type copy of this intron is capable of driving specific seam GFP expression, whereas an intron bearing the two mutations fails to express correctly, suggesting a separate seam-specific role for pal-1. We have demonstrated that the last intron of pal-1 contains an enhancer element (perturbed in e2091) required to correctly specify pal-1 expression in the seam and enable correct seam cell development and orientation. To identify transcription factors that bind to this tissue-specific intronic enhancer we are carrying out a yeast one-hybrid screen using both the wild-type and e2091 mutant version of the pal-1 intron. We will also report our phenotypic analysis of pal-1(e2091) with respect to the cell biology of the seam cells. 1. Edgar, L.G., et al., Dev Biol, 2001. 229(1): p. 71-88. 2. Zhang, H. and S.W. Emmons, Genes Dev, 2000. 14(17): p. 2161-72.

Hunter CP et al. (1995) International C. elegans Meeting "ANCIENT PROTEINS, ANCIENT PATTERNS"

Hunter CP, Kenyon CJ

[International C. elegans Meeting, 1995]

Caudal genes are expressed in posterior cells in both vertebrate and insect early embryos, suggesting that they have a highly conserved role in pattern formation. Antibodies against the C. elegans Caudal homolog pal-1 first detect PAL-1 in the posterior blastomeres EMS and P2 in four-cell embryos. Staining through the 28-cell stage is limited to EMS and P2 progeny. In contrast, pal-1 mRNA appears to be present in all the cells in early embryos. This is very interesting because in Drosophila a posterior gradient of Caudal protein is generated from uniformly distributed mRNA. Thus it is possible that similar mechanisms control early Caudal expression patterns in both cellular C. elegans embryos and syncytial Drosophila embryos. To ask how pal-1 is regulated we have begun to examine PAL-1 expression in mutants defective in early patterning. PAL-1 expression in EMS and P2 is dependent on the par genes but is not dependent on mex-1, which is required for the asymmetric expression of skn-1. This indicates that there are multiple mechanisms for generating asymmetric expression patterns in early embryos. We have also found that pal-1(null) embryos from mosaic mothers that lack pal-1 in the germline die earlier with more severe defects than embryos from heterozygous mothers (previously examined by Edgar & Wood), suggesting a requirement for the maternally expressed pal-1 product.

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.

Hunter CP et al. (1996) West Coast Worm Meeting "THE ACTIVITY OF THE C. ELEGANS CAUDAL HOMOLOG, pal-1, IS SEQUENTIALLY RESTRICTED TO THE POSTERIOR CELLS, C AND D, WHERE IT SPECIFIES CELL IDENTITY"

Hunter CP, Kenyon CJ

[West Coast Worm Meeting, 1996]

The pal-1 protein is homologous to the caudal family of homeodomain proteins. caudal genes are expressed in posterior body regions in all animals examined and function to pattern the posterior body region of Drosophila and mouse. We have found that the pal-1 protein is first detected at the 4-cell stage in the two posterior blastomeres EMS and P2 (Hunter and Kenyon, submitted). However, analysis of embryos from pal-1(-) germline mosaic hermaphrodites shows that pal-1 function is required in only the somatic P2 descendants C and D. These observations raise two questions: How is the pal-1 protein localized to EMS and P2 and how is pal-1 function restricted to P2? We have found that the pal-1 protein distribution pattern is regulated, at least in part, by mex-3 dependent inhibition of translation. mex-3 encodes a putative RNA binding protein that, at the 4-cell stage, is preferentially localized to the anterior blastomeres (Draper et al., submitted). In mex-3 mutant embryos, pal-1 protein is detected in all blastomeres. Furthermore, we found that translation of injected LacZ RNA containing pal-1 3'UTR sequences was inhibited in anterior blastomeres, and that this inhibition was dependent on mex-3 function. In addition, injection of this RNA into wild-type resulted in the production of Mex-3 like embryos; apparently by titrating mex-3 functions. Thus, the pal-1 3' UTR appears to mediate mex-3 dependent regulation of the pal-1 protein expression pattern. pal-1 protein is present in both EMS and P2 yet its function is apparently only required in the somatic descendants of P2. How is pal-1 activity inhibited in EMS? A candidate gene for this inhibitory activity is the Bzip transcription factor skn-1. In skn-1 mutants, EMS cell types are replaced by somatic P2 cell types [Bowerman et al., (1992)]. We injected pal-1 anti-sense RNA into skn-1(-) hermaphrodites and found that these ectopic P2 cell types are dependent on pal-1 function. Thus, skn-1 function in EMS inhibits pal-1 function in EMS. However skn-1 protein is also present in P2, yet it does not appear to inhibit pal-1 function in this cell [Bowerman et al., (1993)]. This suggest that skn-1 cannot inhibit pal-1 activity in P2 or its descendants. A candidate for an inhibitor of skn-1 function in P2 is the pie-1 gene. In pie-1 mutants P2 produces skn-1 dependent EMS-like cell types [Mello et al., (1992)]. These observations suggest a simple model for specifying the identity of EMS and P2 (which will be presented at the meeting because I am out of space here!).

Yurong Yang (2005) International Worm Meeting "pal-1 mRNA distribution is relate to activity of the polarity gene in C.elegans early embryo"

Yurong Yang

[International Worm Meeting, 2005]

A lot of genes involve in C.elegans cell fate determination and linage development. Par genes activities are responsible for establishing anterior and posterior axis. Loss of par function results in loss of AP asymmetries during the first two embryonic cell divisions and a failure to restrict developmental regulators to specific embryonic cells and abnormal cell fate patterning. pal-1 is an important gene which is required to determine the cell fate of posterior blastomeres in C.elegans early embryo. It's a Caudal-like homeodomain protein and specifies the production of body muscle and epidermis by the P2 daugher cells of P1 (Hunter and Keynyon,1996). A lot of genes regulate the expression of PAL-1. Except for mex-3 ，par-1、par-3、par-4 gene also affect the expression of PAL-1 in early embryo as well as the mex-5,mex-6 and spn-4(Nancy N Huang et al 2002). These genes affect the expression and distribution of PAL-1 through regulating the MEX-3 distribution. In order to understand which lever these genes regulate the pattern of PAL-1 expression, we have analyzed the distribution of pal-1 mRNA in C.elegans early embryo of wild type, par-1、par-2、 par-3、par-4、spn-4 mutant and mex-5/mex-6 mutant via in situ hybridization. The observed pal-1 mRNA expression pattern are summarized below: pal-1 mRNA in wild type N2 embryo shows asymmetric localization in one cell, two cell and 4-cell embryos. At 4-cell embryo, posterior 2 cells present high level than anterior cells. In par-1(b274)、par-3(e2074)、par-4(it47) mutant embryos, pal-1mRNA lost asymmetric localization and present everywhere. Loss function of par-2 (it5) does not affect the distribution of pal-1mRNA. At 4-cell embryos, posterior 2 cells present high level than anterior cells, same pattern as wild type embryos. spn-4(or80) and mex-5(zu199) mex-6 (pk440) also affect pal-1mRNA asymmetric distribution. Loss function of spn-4 or mex-5 mex-6, pal-1mRNA lost asymmetric localization and present everywhere. In spn-4 mutant embryo, 2 posterior cells darker staining than 2 anterior cells at 4-cell embryos. In par-1、par-4 mutant, pal-1mRNA present everywhere but no PAL-1 detected in any cells of 4-cell embryos(Bruce Bowerman et al 1997). It implies that pal-1 mRNA distribution is relate to activity of the polarity gene in C.elegans early embryo and the translational regulation of pal-1mRNA may be the reason of spatially and temporally asymmetric location of PAL-1 protein.

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.

Joanne C Wen et al. (2003) International Worm Meeting "Functional Characterization of Early Embryonic PAL-1 Targets"

Craig P Hunter, Joanne C Wen, L Ryan Baugh

[International Worm Meeting, 2003]

The activation and maintenance of C lineage specification occurs through maternal and zygotic PAL-1 activity, respectively (Hunter & Kenyon, 1996; Edgar et al, 2001). A set of targets of this master regulatory transcription factor were identified by transcript profiling embryos with perturbed PAL-1 activity (see abstract by Baugh et al). To functionally characterize PAL-1 targets, we have used RNAi to assess the lethality and terminal phenotypes following loss of function. To identify interactions between targets, we are performing epistasis analysis both by scoring synthetic lethality and by examining the effect of RNAi against one target on the expression of reporters for other targets. Our hope is that such functional characterization of a key set of PAL-1 targets will generate the data necessary to begin modeling the PAL-1 regulatory network.