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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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PHAT-5::MCHERRY localization in wild type and hlh-6 mutants.Fluorescence and NDIC images of (A–C) wild-type and (D–F) hlh-6 animals expressing the hlh-6::phat-5::mCherry translational fusion construct. (B) and (C) are close-ups of animal shown in (A). (E) (F) are close-ups of (D). Fluorescence and NDIC images of (G) wild-type and (K) hlh-6 animals expressing the myo-2::phat-5::mCherry translational fusion construct with corresponding close-ups in (H–I) and (L–M). Arrowheads indicate the pharyngeal lumen, arrows mark the processes of the g1 glands and triangles mark the boundary of the pharyngeal cuticle. PHAT-5::MCHERRY is not found in the intestinal lumen of wild type animals (J) but is present in the intestinal lumen of hlh-6 mutants (N), indicated by carats. Anterior is at left and the pharynx is outlined. Scale bars represent 10 µm.
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pgen-1000222-g008: PHAT-5::MCHERRY localization in wild type and hlh-6 mutants.Fluorescence and NDIC images of (A–C) wild-type and (D–F) hlh-6 animals expressing the hlh-6::phat-5::mCherry translational fusion construct. (B) and (C) are close-ups of animal shown in (A). (E) (F) are close-ups of (D). Fluorescence and NDIC images of (G) wild-type and (K) hlh-6 animals expressing the myo-2::phat-5::mCherry translational fusion construct with corresponding close-ups in (H–I) and (L–M). Arrowheads indicate the pharyngeal lumen, arrows mark the processes of the g1 glands and triangles mark the boundary of the pharyngeal cuticle. PHAT-5::MCHERRY is not found in the intestinal lumen of wild type animals (J) but is present in the intestinal lumen of hlh-6 mutants (N), indicated by carats. Anterior is at left and the pharynx is outlined. Scale bars represent 10 µm.

Mentions: We found that a representative PHAT protein, PHAT-5, lines the pharyngeal lumen, consistent with the protein having a mucin-like function. We examined the subcellular location of PHAT-5 using a phat-5::mCherry fusion expressed under the control of the hlh-6 promoter. The PHAT-5::MCHERRY fusion protein was visible in discrete puncta throughout the cell bodies of the glands, as well as along their extensions (Figure 8A). In live animals, these puncta could be seen to traffic along the extensions, suggesting that the protein had been packaged into secretory vesicles. More importantly, the PHAT-5:: MCHERRY fusion protein was found along the lumen of the pharynx, indicating that the protein had been secreted from the glands (Figure 8A–C). The fusion protein had a discrete anterior boundary, extending as far as the cheilostom groove in the buccal cavity (Figure 8B,C), the boundary between the epidermal cuticle and the pharyngeal cuticle [50], suggesting that PHAT-5 is specifically associated with pharyngeal cuticle. In addition, PHAT-5 fusion protein remained associated with shed pharyngeal cuticle, arguing that the protein forms part of the lining of the pharyngeal lumen (Figure S6). No protein was seen to co-localize with bacteria in the pharynx lumen, suggesting that PHAT-5 does not coat food particles.


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

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

PHAT-5::MCHERRY localization in wild type and hlh-6 mutants.Fluorescence and NDIC images of (A–C) wild-type and (D–F) hlh-6 animals expressing the hlh-6::phat-5::mCherry translational fusion construct. (B) and (C) are close-ups of animal shown in (A). (E) (F) are close-ups of (D). Fluorescence and NDIC images of (G) wild-type and (K) hlh-6 animals expressing the myo-2::phat-5::mCherry translational fusion construct with corresponding close-ups in (H–I) and (L–M). Arrowheads indicate the pharyngeal lumen, arrows mark the processes of the g1 glands and triangles mark the boundary of the pharyngeal cuticle. PHAT-5::MCHERRY is not found in the intestinal lumen of wild type animals (J) but is present in the intestinal lumen of hlh-6 mutants (N), indicated by carats. Anterior is at left and the pharynx is outlined. Scale bars represent 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2563036&req=5

pgen-1000222-g008: PHAT-5::MCHERRY localization in wild type and hlh-6 mutants.Fluorescence and NDIC images of (A–C) wild-type and (D–F) hlh-6 animals expressing the hlh-6::phat-5::mCherry translational fusion construct. (B) and (C) are close-ups of animal shown in (A). (E) (F) are close-ups of (D). Fluorescence and NDIC images of (G) wild-type and (K) hlh-6 animals expressing the myo-2::phat-5::mCherry translational fusion construct with corresponding close-ups in (H–I) and (L–M). Arrowheads indicate the pharyngeal lumen, arrows mark the processes of the g1 glands and triangles mark the boundary of the pharyngeal cuticle. PHAT-5::MCHERRY is not found in the intestinal lumen of wild type animals (J) but is present in the intestinal lumen of hlh-6 mutants (N), indicated by carats. Anterior is at left and the pharynx is outlined. Scale bars represent 10 µm.
Mentions: We found that a representative PHAT protein, PHAT-5, lines the pharyngeal lumen, consistent with the protein having a mucin-like function. We examined the subcellular location of PHAT-5 using a phat-5::mCherry fusion expressed under the control of the hlh-6 promoter. The PHAT-5::MCHERRY fusion protein was visible in discrete puncta throughout the cell bodies of the glands, as well as along their extensions (Figure 8A). In live animals, these puncta could be seen to traffic along the extensions, suggesting that the protein had been packaged into secretory vesicles. More importantly, the PHAT-5:: MCHERRY fusion protein was found along the lumen of the pharynx, indicating that the protein had been secreted from the glands (Figure 8A–C). The fusion protein had a discrete anterior boundary, extending as far as the cheilostom groove in the buccal cavity (Figure 8B,C), the boundary between the epidermal cuticle and the pharyngeal cuticle [50], suggesting that PHAT-5 is specifically associated with pharyngeal cuticle. In addition, PHAT-5 fusion protein remained associated with shed pharyngeal cuticle, arguing that the protein forms part of the lining of the pharyngeal lumen (Figure S6). No protein was seen to co-localize with bacteria in the pharynx lumen, suggesting that PHAT-5 does not coat food particles.

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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