Patterning of dopaminergic neurotransmitter identity among Caenorhabditis elegans ray sensory neurons by a TGFbeta family signaling pathway and a Hox gene

We have investigated the mechanism that patterns dopamine expression among Caenorhabditis elegans male ray sensory neurons. Dopamine is expressed by the A-type sensory neurons in three out of the nine pairs of rays. We used expression of a tyrosine hydroxylase reporter transgene as well as direct assays for dopamine to study the genetic requirements for adoption of the dopaminergic cell fate. In loss-of-function mutants affecting a TGFbeta family signaling pathway, the DBL-1 pathway, dopaminergic identity is adopted irregularly by a wider subset of the rays. Ectopic expression of the pathway ligand, DBL-1, from a heat-shock-driven transgene results in adoption of dopaminergic identity by rays 3-9; rays 1 and 2 are refractory. The rays are therefore prepatterned with respect to their competence to be induced by a DBL-1 pathway signal. Temperature-shift experiments with a temperature-sensitive type II receptor mutant, as well as heat-shock induction experiments, show that the DBL-1 pathway acts during an interval that extends from two to one cell generation before ray neurons are born and begin to differentiate. In a mutant of the AbdominalB class Hox gene egl-5, rays that normally express EGL-5 do not adopt dopaminergic fate and cannot be induced to express DA when DBL-1 is provided by a heat-shock-driven dbl-1 transgene. Therefore, egl-5 is required for making a subset of rays capable of adopting dopaminergic identity, while the function of the DBL-1 pathway signal is to pattern the realization of this capability.     

egl-27 generates anteroposterior patterns of cell fusion in C. elegans by regulating Hox gene expression and Hox protein function.

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.     

High copy arrays containing a sequence upstream of mec-3 alter cell migration and axonal morphology in C. elegans

Background

The Caenorhabditis elegans gene mec-3 encodes a LIM-homeodomain protein that is a master regulator of touch receptor neuron genes. Two of the touch neurons, the ALM neurons, are generated in the anterior of the animal and then migrate to near the middle of the animal. In animals transformed with a sequence upstream of mec-3, the ALM touch receptor neurons failed to migrate to their normal positions and sometimes migrated in the wrong direction, and the PLM touch receptor neurons showed axonal defects. Here we characterize this effect and identify the sequence causing the cell migration and axonal defects.

Results

The ALM migration defect did not result from RNA interference (RNAi), nonspecific effects of carrying a transgenic array, expression of GFP, or the marker gene used to make the transformants. Instead, the ALM migration defect resulted from transgenic arrays containing many copies of a specific 104 bp DNA sequence. Transgenic arrays containing this sequence did not affect all cell migrations.

Conclusions

The mec-3 upstream sequence appeared to be sequestering (titrating out) a specific DNA-binding factor that is required for the ALMs to migrate correctly. Because titration of this factor could reverse the direction of ALM migrations, it may be part of a program that specifies both the direction and extent of ALM migrations. mec-3 is a master regulator of touch receptor neuron genes, so the factor or factors that bind this sequence may also be involved in specifying the fate of touch receptor neurons.     

Pag-3, a Caenorhabditis Elegans Gene Involved in Touch Neuron Gene Expression and Coordinated Movement

Mutations in a newly identified gene, pag-3, cause ectopic expression of touch neuron genes mec-7, mec-7lacZ and mec-4lacZ in the lineal sisters of the ALM touch neurons, the BDU neurons. pag-3 mutants also show a reverse kinker uncoordinated phenotype. The first pag-3 allele was isolated in a screen for mutants with altered immunofluorescence staining patterns. Two additional pag-3 alleles were identified in a noncomplementation screen of 38,000 haploid genomes. All of the pag-3 alleles were recessive to wild type and cause the same phenotypes. Two-factor crosses, deficiency mapping and three-factor crosses located pag-3 to the right arm of the X chromosome between unc-3 and unc-7. Because recessive mutations in pag-3 result in expression of several touch cell specific genes in the BDU neurons, pag-3(+) must directly or indirectly suppress expression of these genes in the BDU neurons. Although pag-3 mutants did not show mec-3lacZ expression in their BDU neurons, expression of mec-7lacZ and mec-4lacZ in the BDU neurons of pag-3 mutants required mec-3(+).     

Patterning of Caenorhabditis elegans posterior structures by the Abdominal-B homolog, egl-5

The Caenorhabditis elegans body axis, like that of other animals, is patterned by the action of Hox genes. In order to examine the function of one C. elegans Hox gene in depth, we determined the postembryonic expression pattern of egl-5, the C. elegans member of the Abdominal-B Hox gene paralog group, by means of whole-mount staining with a polyclonal antibody. A major site of egl-5 expression and function is in the epithelium joining the posterior digestive tract with the external epidermis. Patterning this region and its derived structures is a conserved function of Abd-B paralog group genes in other animals. Cells that initiate egl-5 expression during embryogenesis are clustered around the presumptive anus. Expression is initiated postembryonically in four additional mesodermal and ectodermal cell lineages or tissues. Once initiated in a lineage, egl-5 expression continues throughout development, suggesting that the action of egl-5 can be regarded as defining a positional cell identity. A variety of cross-regulatory interactions between egl-5 and the next more anterior Hox gene, mab-5, help define the expression domains of their respective gene products. In its expression in a localized body region, function as a marker of positional cell identity, and interactions with another Hox gene, egl-5 resembles Hox genes of other animals. This suggests that C. elegans, in spite of its small cell number and reproducible cell lineages, may not differ greatly from other animals in the way it employs Hox genes for regional specification during development.     

Dissection of cis-regulatory elements in the C. elegans Hox gene egl-5 promoter

Hox genes are highly conserved segmental identity genes well known for their complex expression patterns and divergent targets. Here we present an analysis of cis-regulatory elements in the Caenorhabditis elegans Hox gene egl-5, which is expressed in multiple tissues in the posterior region of the nematode. We have utilized phylogenetic footprinting to efficiently identify cis-regulatory elements and have characterized these with gfp reporters and tissue-specific rescue experiments. We have found that the complex expression pattern of egl-5 is the cumulative result of the activities of multiple tissue or local region-specific activator sequences that are conserved both in sequence and near-perfect order in the related nematode Caenorhabditis briggsae. Two conserved regulatory blocks analyzed in detail contain multiple sites for both positively and negatively acting factors. One of these regions may promote activation of egl-5 in certain cells via the Wnt pathway. Positively acting regions are repressed in inappropriate tissues by additional negative pathways acting at other sites within the promoter. Our analysis has allowed us to implicate several new regulatory factors significant to the control of egl-5 expression.     

Regulation of the mec-3 gene by the C.elegans homeoproteins UNC-86 and MEC-3.

The mec-3 gene encodes a homeodomain protein with LIM repeats that is required for the specification of touch cell fate in Caenorhabditis elegans. Previous experiments suggested that mec-3 expression requires the product of the unc-86 gene, a POU-type homeoprotein, and mec-3 itself. We have analyzed the control of mec-3 expression by identifying potential cis regulatory elements in the mec-3 gene (by conservation in a related nematode and by DNase I footprinting using unc-86 and mec-3 proteins) and testing their importance by transforming C.elegans with mec-3lacZ fusions in which these sites have been mutagenized in vitro. Both unc-86 and mec-3 proteins bind specifically to the promoter of the mec-3 gene, suggesting that both proteins may be directly involved in the regulation of the mec-3 gene. In addition, the footprint pattern with mec-3 protein is altered in the presence of unc-86 protein. In vivo transformation experiments reveal that some of the binding regions of the two proteins are needed for general positive control and maintenance of mec-3 expression while others have no detectable, unique function. Interestingly, the unc-86 gene appears to be required not only to initiate mec-3 expression but also to maintain it.     

Protruding vulva mutants identify novel loci and Wnt signaling factors that function during Caenorhabditis elegans vulva development

The Caenorhabditis elegans vulva develops from the progeny of three vulval precursor cells (VPCs) induced to divide and differentiate by a signal from the somatic gonad. Evolutionarily conserved Ras and Notch extracellular signaling pathways are known to function during this process. To identify novel loci acting in vulval development, we carried out a genetic screen for mutants having a protruding-vulva (Pvl) mutant phenotype. Here we report the initial genetic characterization of several novel loci: bar-1, pvl-4, pvl-5, and pvl-6. In addition, on the basis of their Pvl phenotypes, we show that the previously identified genes lin-26, mom-3/mig-14, egl-18, and sem-4 also function during vulval development. Our characterization indicates that (1) pvl-4 and pvl-5 are required for generation/survival of the VPCs; (2) bar-1, mom-3/mig-14, egl-18, and sem-4 play a role in VPC fate specification; (3) lin-26 is required for proper VPC fate execution; and (4) pvl-6 acts during vulval morphogenesis. In addition, two of these genes, bar-1 and mom-3/mig-14, are known to function in processes regulated by Wnt signaling, suggesting that a Wnt signaling pathway is acting during vulval development.     

The T-box gene tbx-2, the homeobox gene egl-5 and the asymmetric cell division gene ham-1 specify neural fate in the HSN/PHB lineage

Understanding how neurons adopt particular fates is a fundamental challenge in developmental neurobiology. To address this issue, we have been studying a Caenorhabditis elegans lineage that produces the HSN motor neuron and the PHB sensory neuron, sister cells produced by the HSN/PHB precursor. We have previously shown that the novel protein HAM-1 controls the asymmetric neuroblast division in this lineage. In this study we examine tbx-2 and egl-5, genes that act in concert with ham-1 to regulate HSN and PHB fate. In screens for mutants with abnormal HSN development, we identified the T-box protein TBX-2 as being important for both HSN and PHB differentiation. TBX-2, along with HAM-1, regulates the migrations of the HSNs and prevents the PHB neurons from adopting an apoptotic fate. The homeobox gene egl-5 has been shown to regulate the migration and later differentiation of the HSN. While mutations that disrupt its function show no obvious role for EGL-5 in PHB development, loss of egl-5 in a ham-1 mutant background leads to PHB differentiation defects. Expression of EGL-5 in the HSN/PHB precursor but not in the PHB neuron suggests that EGL-5 specifies precursor fate. These observations reveal a role for both EGL-5 and TBX-2 in neural fate specification in the HSN/PHB lineage.