Multiple regulatory elements with spatially and temporally distinct activities control the expression of the epithelial differentiation gene lin-26 in C. elegans

Epithelial differentiation is a very early event during development of most species. The nematode Caenorhabditis elegans, with its well-defined and invariant lineage, offers the possibility to link cell lineage, cell fate specification and gene regulation during epithelial differentiation. Here, we focus on the regulation of the gene lin-26, which is required for proper differentiation of epithelial cells in the ectoderm and mesoderm (somatic gonad). lin-26 expression starts in early embryos and remains on throughout development, in many cell types originating from different sublineages. Using GFP reporters and mutant rescue assays, we performed a molecular dissection of the lin-26 promoter and could identify almost all elements required to establish its complex spatial and temporal expression. Most of these elements act redundantly, or synergistically once combined, to drive expression in cells related by function. We also show that lin-26 promoter elements mediate activation in the epidermis (hypodermis) by the GATA factor ELT-1, or repression in the foregut (pharynx) by the FoxA protein PHA-4. Taken together, our data indicate that lin-26 regulation is achieved to a large extent through tissue-specific cis-regulatory elements.     

C. elegans peb-1 mutants exhibit pleiotropic defects in molting, feeding, and morphology

Caenorhabditis elegans PEB-1 is a novel DNA-binding protein expressed in most pharyngeal cell types and outside the pharynx in the hypodermis, hindgut, and vulva. Previous RNAi analyses indicated that PEB-1 is required for normal morphology of these tissues and growth; however, the peb-1 null phenotype was unknown. Here we describe the deletion mutant peb-1(cu9) that not only exhibits the morphological defects observed in peb-1(RNAi) animals, but also results in penetrant larval lethality characterized by defects in pharyngeal function and molting. Consistent with a function in molting, we found that PEB-1 was detectable in all hypodermal and hindgut cells underlying the cuticle. Comparison to molting-defective lrp-1(ku156) mutants revealed that the peb-1(cu9) mutants were particularly defective in shedding the pharyngeal cuticle, and this defect likely contributed to feeding defects and lethality. Most markers of pharyngeal cell differentiation examined were expressed normally in peb-1(cu9) mutants; however, g1 gland cell expression of a kel-1Colon, two colonsgfp reporter was reduced. As g1 gland cells have prominent functions during molting, we suggest defective gland cell differentiation contributes to peb-1(cu9) molting defects. In comparison, other peb-1 mutant phenotypes, including hindgut abnormalities, appeared independent of the molting defect. Similar phenotypes resulted from late loss of pha-4 function, suggesting that PEB-1 and PHA-4 have common functions in some tissues where they are co-expressed.     

Analysis of PHA-1 Reveals a Limited Role in Pharyngeal Development and Novel Functions in Other Tissues

PHA-1 encodes a cytoplasmic protein that is required for embryonic morphogenesis and attachment of the foregut (pharynx) to the mouth (buccal capsule). Previous reports have in some cases suggested that PHA-1 is essential for the differentiation of most or all pharyngeal cell types. By performing mosaic analysis with a recently acquired pha-1 null mutation (tm3671), we found that PHA-1 is not required within most or all pharyngeal cells for their proper specification, differentiation, or function. Rather, our evidence suggests that PHA-1 acts in the arcade or anterior epithelial cells of the pharynx to promote attachment of the pharynx to the future buccal capsule. In addition, PHA-1 appears to be required in the epidermis for embryonic morphogenesis, in the excretory system for osmoregulation, and in the somatic gonad for normal ovulation and fertility. PHA-1 activity is also required within at least a subset of intestinal cells for viability. To better understand the role of PHA-1 in the epidermis, we analyzed several apical junction markers in pha-1(tm3671) homozygous embryos. PHA-1 regulates the expression of several components of two apical junction complexes including AJM-1–DLG-1/discs large complex and the classical cadherin–catenin complex, which may account for the role of PHA-1 in embryonic morphogenesis.     

Coordination of ges-1 expression between the Caenorhabditis pharynx and intestine.

We have previously shown that the Caenorhabditis elegans gut-specific esterase gene (Ce-ges-1) has the unusual ability to be expressed in different modules of the embryonic digestive tract (anterior pharynx, posterior pharynx, and rectum) depending on sequence elements within the Ce-ges-1 promoter. In the present paper, we analyze the expression of the ges-1 homolog (Cb-ges-1) from the related nematode Caenorhabditis briggsae and show that Cb-ges-1 also has the ability to switch expression between gut and pharynx + rectum. The control of this expression switch centres on a tandem pair of WGATAR sites in the Cb-ges-1 5'-flanking region, just as it does in Ce-ges-1. We use sequence alignments and subsequent deletions to identify a region at the 3'-end of both Ce-ges-1 and Ce-ges-1 that acts as the ges-1 cryptic pharynx enhancer whose activity is revealed by removal of the 5' WGATAR sites. This region contains a conserved binding site for PHA-4 (the C. elegans ortholog of forkhead/HNF3 alpha, beta,gamma factors), which is expressed in all cells of the developing pharynx and a subset of cells of the developing rectum. We propose a model in which the normal expression of ges-1 is controlled by the gut-specific GATA factor ELT-2. We propose that, in the pharynx (and rectum), PHA-4 is normally bound to the ges-1 3'-enhancer sequence but that the activation function of PHA-4 is kept repressed by a (presently unknown) factor binding in the vicinity of the 5' WGATAR sites. We suggest that this control circuitry is maintained in Caenorhabditis because pharyngeal expression of ges-1 is advantageous only under certain developmental or environmental conditions.     

NK-2 class homeobox genes and pharyngeal/oral patterning: Nkx2-3 is required for salivary gland and tooth morphogenesis

Head development in vertebrates requires reciprocal patterning interactions between cranial neural crest and the ectodermal, mesodermal and endodermal components of the branchial arches. Patterning elements within the pharyngeal endoderm and oral ectoderm appear to play defining roles in this process. Several homeobox genes of the NK-2 class (Nkx2-1, Nkx2-3, Nkx2-5 and Nkx2-6) are expressed regionally in the developing pharynx, and Nkx2-1 mutants and Nkx2-5/Nkx2-6 double mutants show loss of thyroid and distal lung progenitors, and pharyngeal cell viability, respectively. Here we examined the expression and genetic role of Nkx2-3 in pharyngeal development. Nkx2-3 was expressed in the pharyngeal floor and pouches, as well as in oral and branchial arch ectoderm. Expression persisted in the developing thyroid until birth, in mucous-forming cells of the lingual and sublingual salivary glands, and in odontogenic epithelium of the mandible. Examination of Nkx2-3 null mice revealed defects in maturation and cellular organisation of the sublingual glands. Furthermore, cusps were absent from mandibular molars and the third molar was occasionally missing. These data suggest roles for Nkx2-3 during pharyngeal organogenesis, although the considerable potential for genetic redundancy within and outside of this gene family may mask earlier functions in organ specification.     

The coordinate regulation of pharyngeal development in C. elegans by lin-35/Rb, pha-1, and ubc-18

Organ development is a complex process involving the coordination of cell proliferation, differentiation, and morphogenetic events. Using a screen to identify genes that function coordinately with lin-35/Rb during animal development, we have isolated a weak loss-of-function (LOF) mutation in pha-1. lin-35; pha-1 double mutants are defective at an early step in pharyngeal morphogenesis leading to an abnormal pharyngeal architecture. pha-1 is also synthetically lethal with other class B synthetic multivulval (SynMuv) genes including the C. elegans E2F homolog, efl-1. Reporter analyses indicate that pha-1 is broadly expressed during embryonic development and that its functions reside in the cytoplasm. We also provide genetic and phenotypic evidence to support the model that PHA-1, a novel protein, and UBC-18, a ubiquitin-conjugating enzyme that we have previously shown to function with lin-35 during pharyngeal development, act in parallel pathways to regulate the activity of a common cellular target.