Conserved regulation of the Caenorhabditis elegans labial/Hox1 gene ceh-13

Caenorhabditis elegans contains a set of six cluster-type homeobox (Hox) genes that are required during larval development. Some of them, but unlike in flies not all of them, are also required during embryogenesis. It has been suggested that the control of the embryonic expression of the worm Hox genes might differ from that of other species by being regulated in a lineal rather than a regional mode. Here, we present a trans-species analysis of the cis-regulatory region of ceh-13, the worm ortholog of the Drosophila labial and the vertebrate Hox1 genes, and find that the molecular mechanisms that regulate its expression may be similar to what has been found in species that follow a regulative, non-cell-autonomous mode of development. We have identified two enhancer fragments that are involved in different aspects of the embryonic ceh-13 expression pattern. We show that important features of comma-stage expression depend on an autoregulatory input that requires ceh-13 and ceh-20 functions. Our data show that the molecular nature of Hox1 class gene autoregulation has been conserved between worms, flies, and vertebrates. The second regulatory sequence is sufficient to drive correct early embryonic expression of ceh-13. Interestingly, this enhancer fragment acts as a response element of the Wnt/WG signaling pathway in Drosophila embryos.     

pha-2 encodes the C. elegans ortholog of the homeodomain protein HEX and is required for the formation of the pharyngeal isthmus

The pha-2 mutant was isolated in 1993 by Leon Avery in a screen for worms with visible defects in pharyngeal feeding behavior. In pha-2 mutant worms, the pharyngeal isthmus is abnormally thick and short and, in contrast to wild-type worms, harbors several cell nuclei. We show here that pha-2 encodes a homeodomain protein and is homologous to the vertebrate homeobox gene, Hex (also known as Prh). Consistent with a function in pharyngeal development, the pha-2 gene is expressed in the pharyngeal primordium of Caenorhabditis elegans embryos, particularly in pm5 cells that form the bulk of the isthmus. We show that in the pha-2 mutant there is a failure of the pm5 cells to elongate anteriorly while keeping their nuclei within the nascent posterior bulb to form the isthmus during the 3-fold embryonic stage. We also present evidence that pha-2 regulates itself positively in pm5 cells, that it is a downstream target of the forkhead gene pha-4, and that it may also act in the isthmus as an inhibitor of the ceh-22 gene, an Nkx2.5 homolog. Finally, we have begun characterizing the regulation of the pha-2 gene and find that intronic sequences are essential for the complete pha-2 expression profile. The present report is the first to examine the expression and function of an invertebrate Hex homolog, that is, the C. elegans pha-2 gene.     

Misexpression of acetylcholinesterases in the C. elegans pha-2 mutant accompanies ultrastructural defects in pharyngeal muscle cells

pha-2 is the Caenorhabditis elegans homolog of the vertebrate homeobox gene Hex. Embryonic expression of pha-2 is mostly pharyngeal and the only described mutant allele of pha-2 results in a severe pharyngeal defect in which certain muscle cells (pm5 cells) and neurons are grossly deformed. Here, we performed a detailed characterization of the pha-2 phenotype using cell-type-specific reporters, physical manipulation of the nuclei in pharyngeal muscle cells using "optical tweezers", electron microscopy, staining of the actin cytoskeleton as well as phenotypic rescue and ectopic expression experiments. The main findings of the present study are (i) the pha-2 (ad472) mutation specifically impairs the pharyngeal expression of pha-2; (ii) in the pha-2 mutant, the cytoskeleton of the pm5 cells is measurably weaker than in normal cells and is severely disrupted by large tubular structures and organelles; (iii) the pm5 cells of the pha-2 mutant fail to express the acetylcholinesterase genes ace-1 and ace-2; (iv) ectopic expression of pha-2 can induce ectopic expression of ace-1 and ace-2; and (v) the anc-1 mutant with mislocalized pm5 cell nuclei occasionally shows an isthmus phenotype similar to that of pha-2 worms.     

Behavioral and synaptic defects in C. elegans lacking the NK-2 homeobox gene ceh-28

C. elegans pharyngeal behavior consists of two distinct types of muscle contractions, termed pumping and peristalsis. Pumping ingests and concentrates bacteria in the anterior pharyngeal lumen, and it is occasionally followed by a transient peristaltic contraction that carries ingested bacteria through the posterior pharyngeal isthmus. These behaviors are controlled by a small pharyngeal nervous system consisting of 20 neurons that is almost completely independent of the extra-pharyngeal nervous system. The cholinergic motor neuron M4 controls peristalsis via synapses with the posterior isthmus muscles. Here we show that the NK-2 family homeobox gene ceh-28 is expressed in M4, where it regulates synapse assembly and peristalsis. ceh-28 mutants exhibit frequent and prolonged peristalses, and treatment with agonists or antagonists of muscarinic acetylcholine receptors can phenocopy or suppress ceh-28 mutant defects, respectively. Synapses in ceh-28 mutant M4 cells are irregularly spaced and sized, and they are abnormally located along the full length of the isthmus. We suggest that CEH-28 inhibits synaptogenesis, and that ceh-28 mutant behavioral defects result from excessive or ectopic stimulation of muscarinic acetylcholine receptors in the isthmus muscles.     

Calcineurin regulates enteric muscle contraction through EXP-1, excitatory GABA-gated channel, in C. elegans

The enteric muscle contraction (EMC) is the last step of the defecation behavior which occurs every 50 s in Caenorhabditis elegans. This EMC is regulated by intestinal and anal depressor muscles, which are innervated by GABA motor neurons. Our data show that calcineurin (tax-6) is expressed in intestinal muscle and anal depressor muscle, and the gain-of-function mutant of calcineurin, tax-6(jh107), shows defects in enteric muscle contractions. In addition, the intracellular region of EXP-1, an excitatory GABA receptor, specifically binds to calcineurin A. This interaction between TAX-6 and EXP-1 appears to be independent of both calcium and CNB, which is the calcium-binding regulatory subunit. Genetic evidence of epistasis between cnb-1(jh103) and exp-1(sa6) suggests that calcineurin functions as a negative regulator of excitatory GABA receptor in GABA signaling in C.elegans.     

SER-1, a Caenorhabditis elegans 5-HT2-like receptor, and a multi-PDZ domain containing protein (MPZ-1) interact in vulval muscle to facilitate serotonin-stimulated egg-laying

Serotonin (5-HT) stimulation of egg-laying in Caenorhabditis elegans is abolished in ser-1 (ok345) animals and is rescued by ser-1 expression in vulval muscle. A PDZ binding motif (ETFL) at the SER-1 C-terminus is not essential for rescue, but facilitates SER-1 signaling. SER-1 binds specifically to PDZ domain 10 of the multi-PDZ domain protein, MPZ-1, based on GST pulldown and co-immunoprecipitation. mpz-1 is expressed in about 60 neurons and body wall and vulval muscles. In neurons, GFP-tagged MPZ-1 is punctate and colocalizes with the synaptic marker, synaptobrevin. The expression patterns of ser-1 and mpz-1 overlap in 3 pairs of neurons and vulval muscle. In addition, MPZ-1 also interacts with other GPCRs with acidic amino acids in the -3 position of their PDZ binding motifs. mpz-1 RNAi reduces 5-HT stimulated egg-laying in wild type animals and in ser-1 mutants rescued by muscle expression of SER-1. In contrast, mpz-1 RNAi has no effect on 5-HT stimulated egg-laying in ser-1 mutants rescued by expression of a truncated SER-1 that lacks the C-terminal PDZ binding motif. The overexpression of MPZ-1 PDZ domain 10 also inhibits 5-HT stimulated egg-laying. These studies suggest that the SER-1/MPZ-1 interaction facilitates SER-1 mediated signaling.     

Cre-mediated excision of Fgf8 in the Tbx1 expression domain reveals a critical role for Fgf8 in cardiovascular development in the mouse

Tbx1 has been implicated as a candidate gene responsible for defective pharyngeal arch remodeling in DiGeorge/Velocardiofacial syndrome. Tbx1(+/-) mice mimic aspects of the DiGeorge phenotype with variable penetrance, and null mice display severe pharyngeal hypoplasia. Here, we identify enhancer elements in the Tbx1 gene that are conserved through evolution and mediate tissue-specific expression. We describe the generation of transgenic mice that utilize these enhancer elements to direct Cre recombinase expression in endogenous Tbx1 expression domains. We use these Tbx1-Cre mice to fate map Tbx1-expressing precursors and identify broad regions of mesoderm, including early cardiac mesoderm, which are derived from Tbx1-expressing cells. We test the hypothesis that fibroblast growth factor 8 (Fgf8) functions downstream of Tbx1 by performing tissue-specific inactivation of Fgf8 using Tbx1-Cre mice. Resulting newborn mice display DiGeorge-like congenital cardiovascular defects that involve the outflow tract of the heart. Vascular smooth muscle differentiation in the great vessels is disrupted. This data is consistent with a model in which Tbx1 induces Fgf8 expression in the pharyngeal endoderm, which is subsequently required for normal cardiovascular morphogenesis and smooth muscle differentiation in the aorta and pulmonary artery.     

Two Hox cofactors, the Meis/Hth homolog UNC-62 and the Pbx/Exd homolog CEH-20, function together during C. elegans postembryonic mesodermal development

The TALE homeodomain-containing PBC and MEIS proteins play multiple roles during metazoan development. Mutations in these proteins can cause various disorders, including cancer. In this study, we examined the roles of MEIS proteins in mesoderm development in C. elegans using the postembryonic mesodermal M lineage as a model system. We found that the MEIS protein UNC-62 plays essential roles in regulating cell fate specification and differentiation in the M lineage. Furthermore, UNC-62 appears to function together with the PBC protein CEH-20 in regulating these processes. Both unc-62 and ceh-20 have overlapping expression patterns within and outside of the M lineage, and they share physical and regulatory interactions. In particular, we found that ceh-20 is genetically required for the promoter activity of unc-62, providing evidence for another layer of regulatory interactions between MEIS and PBC proteins.