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The HLH-6 transcription factor regulates C. elegans pharyngeal gland development and function.

Smit RB, Schnabel R, Gaudet J - PLoS Genet. (2008)

Bottom Line: Interestingly, hlh-6 mutants are also feeding defective, ascribing a biological function for the glands.Pharyngeal pumping in hlh-6 mutants is normal, but hlh-6 mutants lack expression of a class of mucin-related proteins that are normally secreted by pharyngeal glands and line the pharyngeal cuticle.An interesting possibility is that one function of pharyngeal glands is to secrete a pharyngeal lining that ensures efficient transport of food along the pharyngeal lumen.

View Article: PubMed Central - PubMed

Affiliation: Genes and Development Research Group, Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT
The Caenorhabditis elegans pharynx (or foregut) functions as a pump that draws in food (bacteria) from the environment. While the "organ identity factor" PHA-4 is critical for formation of the C. elegans pharynx as a whole, little is known about the specification of distinct cell types within the pharynx. Here, we use a combination of bioinformatics, molecular biology, and genetics to identify a helix-loop-helix transcription factor (HLH-6) as a critical regulator of pharyngeal gland development. HLH-6 is required for expression of a number of gland-specific genes, acting through a discrete cis-regulatory element named PGM1 (Pharyngeal Gland Motif 1). hlh-6 mutants exhibit a frequent loss of a subset of glands, while the remaining glands have impaired activity, indicating a role for hlh-6 in both gland development and function. Interestingly, hlh-6 mutants are also feeding defective, ascribing a biological function for the glands. Pharyngeal pumping in hlh-6 mutants is normal, but hlh-6 mutants lack expression of a class of mucin-related proteins that are normally secreted by pharyngeal glands and line the pharyngeal cuticle. An interesting possibility is that one function of pharyngeal glands is to secrete a pharyngeal lining that ensures efficient transport of food along the pharyngeal lumen.

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The extended PGM1 is sufficient for gland-specific expression.(A) Alignment of PGM1 occurrences in the promoters of gland-expressed genes. Expression of genes in bold is experimentally verified to be both PGM1 and HLH-6 dependent. (B–E) Fluorescence micrographs of GFP enhancer constructs containing (B) no insert, (C) three tandem copies of the extended PGM1, (D) three tandem copies of the extended PGM1 in which the E-box has been mutated and (E) three tandem copies of the extended PGM1 in which sequence flanking the E-box has been altered. Anterior is at left and the pharynx is outlined. Scale bars represent 10 µm.
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pgen-1000222-g002: The extended PGM1 is sufficient for gland-specific expression.(A) Alignment of PGM1 occurrences in the promoters of gland-expressed genes. Expression of genes in bold is experimentally verified to be both PGM1 and HLH-6 dependent. (B–E) Fluorescence micrographs of GFP enhancer constructs containing (B) no insert, (C) three tandem copies of the extended PGM1, (D) three tandem copies of the extended PGM1 in which the E-box has been mutated and (E) three tandem copies of the extended PGM1 in which sequence flanking the E-box has been altered. Anterior is at left and the pharynx is outlined. Scale bars represent 10 µm.

Mentions: Closer examination of PGM1 revealed that it contains an E-box (CAnnTG), the consensus binding site for basic helix-loop-helix (bHLH) transcription factors [23]. Mutations that specifically disrupt the E-box sequence eliminate PGM1 activity (Figure 1). However, the E-box is not sufficient for PGM1 activity: mutation of sequence flanking the E-box in the phat-1 reporter resulted in a significant loss of expression (data not shown), suggesting that an extended sequence is required for activity. Alignment of the functionally defined PGM1 sequences revealed an extended consensus of CAnvTGhdYMAAY (where V = A, C or G, H = A, C or T, D = A, G or T, M = A or C, and Y = C or T; Figure 2A). This extended consensus is present in all 12 of the 14 genes in our initial list that contained PGM1 (Figure 2A). The functionally defined consensus may represent either an extended binding preference for the relevant trans-acting factor or the juxtaposition of binding sites for two (or more) distinct factors.


The HLH-6 transcription factor regulates C. elegans pharyngeal gland development and function.

Smit RB, Schnabel R, Gaudet J - PLoS Genet. (2008)

The extended PGM1 is sufficient for gland-specific expression.(A) Alignment of PGM1 occurrences in the promoters of gland-expressed genes. Expression of genes in bold is experimentally verified to be both PGM1 and HLH-6 dependent. (B–E) Fluorescence micrographs of GFP enhancer constructs containing (B) no insert, (C) three tandem copies of the extended PGM1, (D) three tandem copies of the extended PGM1 in which the E-box has been mutated and (E) three tandem copies of the extended PGM1 in which sequence flanking the E-box has been altered. Anterior is at left and the pharynx is outlined. Scale bars represent 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2563036&req=5

pgen-1000222-g002: The extended PGM1 is sufficient for gland-specific expression.(A) Alignment of PGM1 occurrences in the promoters of gland-expressed genes. Expression of genes in bold is experimentally verified to be both PGM1 and HLH-6 dependent. (B–E) Fluorescence micrographs of GFP enhancer constructs containing (B) no insert, (C) three tandem copies of the extended PGM1, (D) three tandem copies of the extended PGM1 in which the E-box has been mutated and (E) three tandem copies of the extended PGM1 in which sequence flanking the E-box has been altered. Anterior is at left and the pharynx is outlined. Scale bars represent 10 µm.
Mentions: Closer examination of PGM1 revealed that it contains an E-box (CAnnTG), the consensus binding site for basic helix-loop-helix (bHLH) transcription factors [23]. Mutations that specifically disrupt the E-box sequence eliminate PGM1 activity (Figure 1). However, the E-box is not sufficient for PGM1 activity: mutation of sequence flanking the E-box in the phat-1 reporter resulted in a significant loss of expression (data not shown), suggesting that an extended sequence is required for activity. Alignment of the functionally defined PGM1 sequences revealed an extended consensus of CAnvTGhdYMAAY (where V = A, C or G, H = A, C or T, D = A, G or T, M = A or C, and Y = C or T; Figure 2A). This extended consensus is present in all 12 of the 14 genes in our initial list that contained PGM1 (Figure 2A). The functionally defined consensus may represent either an extended binding preference for the relevant trans-acting factor or the juxtaposition of binding sites for two (or more) distinct factors.

Bottom Line: Interestingly, hlh-6 mutants are also feeding defective, ascribing a biological function for the glands.Pharyngeal pumping in hlh-6 mutants is normal, but hlh-6 mutants lack expression of a class of mucin-related proteins that are normally secreted by pharyngeal glands and line the pharyngeal cuticle.An interesting possibility is that one function of pharyngeal glands is to secrete a pharyngeal lining that ensures efficient transport of food along the pharyngeal lumen.

View Article: PubMed Central - PubMed

Affiliation: Genes and Development Research Group, Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT
The Caenorhabditis elegans pharynx (or foregut) functions as a pump that draws in food (bacteria) from the environment. While the "organ identity factor" PHA-4 is critical for formation of the C. elegans pharynx as a whole, little is known about the specification of distinct cell types within the pharynx. Here, we use a combination of bioinformatics, molecular biology, and genetics to identify a helix-loop-helix transcription factor (HLH-6) as a critical regulator of pharyngeal gland development. HLH-6 is required for expression of a number of gland-specific genes, acting through a discrete cis-regulatory element named PGM1 (Pharyngeal Gland Motif 1). hlh-6 mutants exhibit a frequent loss of a subset of glands, while the remaining glands have impaired activity, indicating a role for hlh-6 in both gland development and function. Interestingly, hlh-6 mutants are also feeding defective, ascribing a biological function for the glands. Pharyngeal pumping in hlh-6 mutants is normal, but hlh-6 mutants lack expression of a class of mucin-related proteins that are normally secreted by pharyngeal glands and line the pharyngeal cuticle. An interesting possibility is that one function of pharyngeal glands is to secrete a pharyngeal lining that ensures efficient transport of food along the pharyngeal lumen.

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