The C. elegans gene pan-1 encodes novel transmembrane and cytoplasmic leucine-rich repeat proteins and promotes molting and the larva to adult transition

Abstract

Background: Extracellular leucine-rich repeat (eLRR) proteins are a highly diverse superfamily of membrane-associated or secreted proteins. In the membrane-associated eLRR proteins, the leucine-rich repeat motifs interact with the extracellular matrix and other ligands. Characterizing their functions in animal model systems is key to deciphering their activities in various developmental processes.

Results: In this study, we identify pan-1 as a critical regulator of C. elegans larval development. pan-1 encodes both transmembrane and cytoplasmic isoforms that vary in the presence and number of leucine-rich repeats. RNAi experiments reveal that pan-1 is required for developmental processes that occur during the mid to late larval stages. Specifically, pan-1 loss of function causes a late larval arrest with a failure to complete development of the gonad, vulva, and hypodermis. pan-1 is also required for early larval ecdysis and execution of the molting cycle at the adult molt. We also provide evidence that pan-1 functionally interacts with the heterochronic gene lin-29 during the molting process.

Conclusions: We show that PAN-1 is a critical regulator of larval development. Our data suggests that PAN-1 promotes developmental progression of multiple tissues during the transition from a larva to a reproductive adult. We further demonstrate that the activity of PAN-1 is complex with diverse roles in the regulation of animal development.

Figure 1

pan-1 gene structure and mRNA isoform expression. (A) mRNA splicing pattern of pan-1 and predicted encoded proteins. RT-PCR data indicate three SL1 trans-splice sites that would generate three distinct mRNA isoforms: pan-1A, pan-1B, and pan-1C. The isoforms generated by the SL1 trans-splice sites are indicated in parentheses. Possible translational start codons for the different isoforms are also indicated. The encoded PAN-1B protein isoform has 11 LRRs (vertical stippled boxes) as identified by the SMART program. An additional 4 LRRs are predicted by LRRscan, as described in Dolan et al. 2007 (yellow boxes). The N and C terminal LRR capping domains (blue colored regions) were identified using the consensus sequence found in Dolan et al., 2007. The PAN-1 N-terminal capping domain (LRR-NT) has the amino sequence CIDIEKGFKEEFNAHKQP VCICADNGIFSTVKGFTIEC. The C-terminal PAN-1 capping domain (LRR-CT1) has the amino acid sequence PWVCVCNDPKEWLPRWLEASEEADVAEGALGCLAIPNC. The signal peptide (‘SP’) is indicated by the red colored region and the transmembrane domain (‘TM’) is indicated by the purple colored region. The cytoplasmic domain (‘CD’) and GFP fusion point (green bar) are also indicated. PAN-1A is not predicted to encode a transmembrane protein and contains fewer LRRs. (B) RT-PCR analysis of pan-1 on total RNA isolated from glp-1(bn2ts) and also wild-type adults subjected to control or pan-1 RNAi (1% agarose gel stained with ethidium bromide). glp-1(bn2ts) larvae raised to adulthood at 16°C form a germline while animals raised at 25°C lack germline tissue. pan-1A and pan-1B are amplified from cDNA generated from both populations while pan-1C is only robustly amplified from cDNA generated from animals raised at 16°C, indicating germline enrichment of pan-1C relative to the soma. All three isoforms are also amplified from cDNA generated from wild-type adult animals. unc-54 is a somatic gene that is expressed in both 16°C and 25°C cDNA preparations.

Figure 2

pan-1 is required for larval development. (A) Graph showing the percentages of larval arrest phenotypes in pan-1 and control RNAi experiments. L4prg and L4ecd larval arrest phenotypes are differentiated by a ‘clear’ appearance and un-molted pharyngeal cuticle in the L4ecd larvae. L4prg larvae appear healthy and are viable. (B, C) Representative micrographs of time-matched pan-1 and control RNAi hermaphrodites (wild-type background). pan-1(RNAi) L4prg hermaphrodite is shown in (B). L4prg larvae are smaller than control animals (C) and sterile (no eggs generated). Scale bars = 100 μm (D) An arrested pan-1(RNAi) L3 larva encased in L2 cuticle (arrow). (E) An arrested L4prg hermaphrodite partially encased in the posterior segment of L3 cuticle (arrow). Posterior is to the right. Scale bars = 10 μm.

Figure 3

Gonadal growth and vulva development phenotypes of pan-1(RNAi) hermaphrodites. (A) Graph showing the gonadal growth scores of pan-1 and control RNAi animals. The number of animals scored for each experiment is indicated. Also shown is RT-PCR analysis of rrf-1(pk1417) cultures (harvested at mid-L4 stage) subjected to control or pan-1RNAi. DIC micrographs of control RNAi (B, C) and pan-1 RNAi (D–G) animals. In images (B-G), the thin arrow indicates vulva, thick arrow indicates distal tip of gonad, and asterisk developing gametes. (B) Control RNAi hermaphrodite at 54 hrs post-feeding. (C) Control RNAi mid-L4 hermaphrodite (38–40 hrs post-feeding). Note luminal stage of vulval development and degree of gonadal growth. (D)pan-1(RNAi) hermaphrodite at 54 hrs post-feeding exhibiting gonadal growth score of 1 and arrested vulva development at the luminal stage. (E)pan-1(RNAi) hermaphrodite at 54 hrs post-feeding with a gonadal growth score of 3 (distal end not shown) and producing gametes. Vulva remains at the luminal stage of development. (F)rrf-1(−); pan-1(RNAi) adult hermaphrodite exhibiting a normal vulval morphology and a morphologically abnormal but differentiated somatic gonad. (G)rrf-1(−); pan-1(RNAi) adult hermaphrodite exhibiting a protruding vulva, a gonadal growth score of 1 and absence of somatic gonad tissue. Both of the animals in (F) and (G) completed the L4 to adult molt and synthesized lateral alae. Epifluorescence (H-J) and corresponding DIC micrographs of AJM-1:GFP expression in control (H) and pan-1 RNAi hermaphrodites (I, J). The animal in (I) exhibits an absence of spermathecal tissue (thick arrow) while the animal in (J) exhibits an abnormally formed and small spermatheca. Epifluorescence micrographs (K, L) of nuclear localized NHR-6:GFP expression in control (K) and pan-1 RNAi hermaphrodites (L). Normal spermathecae in late larvae and adults have 24 cells (thick arrow). In the pan-1(RNAi) animal one gonad arm has two expressing nuclei while the other has a single expressing nucleus. Scale bars = 10 μm.

Figure 4

Hypodermal defects of arrested pan-1(RNAi) animals. Epifluorescence micrographs of ajm-1::GFP(A, B) and col-19::GFP(C, D) in control (A, C) and pan-1 RNAi (B, D) animals. For (A) and (B), the areas marked by the blue box are shown as enlarged DIC images (A’ and B’). Animals subjected to pan-1 RNAi do not synthesize alae (arrows in A’) in areas of normal seam fusion. The arrows in (B) represent areas of abnormal seam morphology including lack of fusion and abnormal morphology (arrow marked with an asterisk). (C) and (D)col-19::GFP is not expressed in arrested pan-1(RNAi) animals. (E) Quantification of seam phenotypes in control (red bar) and pan-1 (blue bar) RNAi animals. The stippling indicates the proportion of animals exhibiting completely normal seam morphology. N = 41 for pan-1 RNAi; N = 20 for control RNAi. Scale bars = 10 μm.

Figure 5

Expression of mlt-10::GFP. Percentage of animals expressing mlt-10::GFP at the L3/L4 transition (38-41hrs post-feeding) and L4/Adult transition (48–51 hrs post-feeding). N = 30 and 25 for control RNAi animals at L3/L4 and L4/Adult, respectively, and N = 33 and 36, respectively, for pan-1 RNAi animals.

Figure 6

pan-1::GFP expression pattern. (A) Expression of pan-1prom::GFP in the somatic gonad and vulva. The proximal sheath (Sh), spermatheca (Sp), and uterus (Ut) and vulva are indicated. (B)pan-1prom::GFP expression in body wall muscle. Arrow indicates muscle cell nucleus. (C) A merged epiflourescence and DIC micrograph showing expression of PAN-1FL::GFP in the spermatheca, uterus, and vulva. The arrows indicate PAN-1FL::GFP punctae in the apical (lumenal surface) regions of spermatheca and uterine epithelia. (D) A ventral view of a developing L4 stage vulva showing PAN-1FL::GFP punctae clustered on the apical ends of vulva epithelial cells. Cytoplasmic fluorescence is also observed in these cells (E) PAN-1FL::GFP in a seam cell. Arrow indicates a punctate region of localization and cytoplasmic fluorescence is also observed. (F) Expression of PAN-1FL::GFP in the pharynx and anterior hypodermis. Cytoplasmic fluorescence is also observed in the pharyngeal tissue. Arrow indicates punctate localization in the hypodermis. Immunolocalization of MH27 (G) and PAN-1FL::GFP (H) in the developing pharyngeal epithelium. PAN-1FL::GFP is found in the developing pharyngeal epithelium (arrow) of a bean stage embryo. (I) Merged image of (G) and (H). Scale bars = 10 μm.

Figure 7

pan-1 RNAi interactions with lin-29 and let-7. (A)lin-29 (−); pan-1(RNAi) animal with a pronounced Pvl phenotype (arrow). (B)lin-29 (−); pan-1(RNAi) animal with a smaller protruding vulva. Both animals in (A) and (B) exhibit an L3 gonadal arrest (*) and the animal in (B) is encased in unshed cuticle (arrowheads). (C)let-7(−); pan-1(RNAi) animals with an L4 arrest phenotype marked by the developmentally arrested vulva (arrow) and stunted gonad (*). (D) The percentage of animals exhibiting the “exploded out of the vulva” phenotype. The let-7 experiments were performed at 25°C as the let-7(n2853) allele is temperature-sensitive. The L4 arrest and exploded phenotype were scored at 74 hrs post-feeding. N = 126 for pan-1 RNAi and N = 98 for control RNAi. Scale bars = 10 μm.