The HLH-6 transcription factor regulates C. elegans pharyngeal gland development and function - PubMed (original) (raw)

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

Ryan B Smit et al. PLoS Genet. 2008 Oct.

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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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. PGM1 is required for expression of some pharyngeal gland genes.

(A) Diagram of pharynx, highlighting the pharyngeal glands, modified from . (B) WebLogo of computationally identified PGM1. (C–N) Fluorescence micrographs of gland-expressed GFP or YFP reporters with wild-type promoter sequence (left column) or promoter sequence in which PGM1 is mutated (right column). In wild-type sequences (left) the E-box is underlined and in mutant sequences (right) the mutation is underlined. Anterior is at left and the pharynx is outlined. Scale bars represent 10 µm.

Figure 2

Figure 2. 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.

Figure 3

Figure 3. hlh-6 is expressed in pharyngeal glands.

(A) Schematic of the genomic region containing hlh-6. The position of the deletion allele tm299 is indicated. The portion of hlh-6 encoding the DNA Binding Domain (DBD) is shown as is the hlh-6 “minigene”, which rescues all aspects of the hlh-6 mutant phenotype. (B) Expression of the hlh-6::YFP reporter, containing 1175 bp (of 1190 bp) of intergenic sequence from the ATG of hlh-6 to just downstream of the stop codon of the next upstream gene, T15H9.2. Anterior is at left and the pharynx is outlined. Scale bar represents 10 µm.

Figure 4

Figure 4. hlh-6 is required for PGM1 activity.

(A) An example of hlh-6 mutants where expression of phat-1::YFP is not visible. (B) An example of hlh-6 mutants where expression of hlh-6::YFP is visible only in g1 cells. The absence of the g2 cells is indicated by the arrow. Anterior is at left and the pharynx is outlined. Scale bars represent 10 µm. (C) Quantitation of the number of animals expressing each reporter in hlh-6 mutants. For the phat-1::YFP reporter in wildtype and hlh-6 mutants, only one transgenic line was scored but the same array was used in both genotypes. Two lines of the genomic rescue were scored for phat-1::YFP expression (lines 5 and 2). Only one line of minigene rescue was scored. Number of animals scored is indicated.

Figure 5

Figure 5. The g2 glands are not generated in hlh-6 mutants.

The lineages of the g2 glands in wild-type and hlh-6 mutants. MSn is used because both the MSa and MSp cell give rise to a g2 cell. If n = a, the g2L cell is made (as well as pm6VL and vpi2DL) and if n = p, the g2R cell is made (and pm6VR and vpi2DR). The sister cell of g2 cell undergoes apoptosis (X) in wild-type animals.

Figure 6

Figure 6. Phenotypic analysis of hlh-6 mutants.

(A–B) The stuffed pharynx phenotype of hlh-6 mutants grown on OP50-GFP bacteria. (A) NDIC image, (B) merged NDIC and fluorescence image. Anterior is at left and scale bars represent 10 µm. (C–E) Assays for growth defects in wild-type, hlh-6 mutants and hlh-6 mutants rescued by either the hlh-6 genomic fragment, the hlh-6 minigene or by using the HB101 strain of E. coli. (C) Graph of body length versus time, (D) time to reach adulthood and (E) brood sizes. For the hlh-6 mutants the L1 arrested animals are omitted. Error bars represent one standard deviation.

Figure 7

Figure 7. Staining of intestinal fat stores of hlh-6 mutants.

(A–D) Fluorescence images of animals grown in the presence of Nile Red. (A) wild type, (B) daf-16(RNAi), (C) hlh-6 and (D) hlh-6; daf-16(RNAi). Anterior is at left and the pharynx is outlined. Scale bars represent 10 µm.

Figure 8

Figure 8. 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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