PQBP1 Affects Lipid Storage Figure 1. Construction of transgenic C. elegans expressing Venus under the control of T21D12.3 enhancer/promoter. T21D12.3 and T21D12.4 (pat-6) are transcriptionally regulated as an operon. Therefore, 6.7 kb genome DNA from the transcriptional initiation site to the neighboring gene to pat-6 was subcloned into pGEMT-T plasmid. By injecting the plasmid into germ line of adult worm, we obtained transgenic nematodes in which the Venus cDNA was subcloned into exon 4 to express a PQBP1-Venus fusion protein under the control of endogenous enhancer/promoter. doi:10.1371/journal.pone.0004104.g001 By injecting the reporter plasmid, we obtained F2 transgenic worms as described in Materials and Methods. The fluorescence signals of the pqbp-1.1-Venus fusion protein indicated that pqbp-1.1 was expressed from 24-cells stage to Larva stage 4 (L4) (Fig. 2, 3). From 24 cells- to gastrulation-stage, pqbp-1.1-Venus was expressed ubiquitously (Fig. 2). At L1 stage, neural cells in the head ganglion express the fusion protein (Fig. 3A). From late L1 stage, intestinal cells expressed pqbp-1.1-Venus strongly and the high expression continued to L4 stage. The peak of expression was observed at L3 stage. Pharyngeal cells show an almost similar pattern of expression during development, while the peak seems to be at L4 stage (Fig. 3C). Higher magnification of the signals revealed that the fusion protein formed speckle-like intranuclear dots (or nuclear bodies) just like mammalian PQBP1 [2,11] (Fig. 3B, 3C). After becoming adult worm, pqbp-1.1-Venus expression reduced rapidly in pharynx, while the expression continued in intestinal cells (Fig. 4). PQBP1 expression in somatic gonads and head ganglions During the development, somatic gonad cells expressed pqbp- 1.1-Venus only from L3 to L4 stage (Fig. 3B, C), and adult gonads or fertilized eggs did not express pqbp-1.1-Venus (Fig. 4). Pqbp-1.1 expression was also observed in head ganglion cells at L1 stage. The expression in such neural precursor cells is consistent with the developmental expression pattern of PQBP1 in mice [14]. Some neurons in the tail show weak expression of pqbp-1.1-Venus in adult worms (Fig. 4). However, after L1 stage, expression in neurons was relatively weak in comparison to that in intestinal cells or somatic gonad, and further analysis is needed to clarify the detail. Depletion of PQBP1 homologue leads to reduction of lipid storage Screening of the T21D12.3 mutants was performed as described previously [15]. Consequently, a mutant (tm3004) was obtained from the screening, which lacks a part of the T21D12.3 genome including the first methionine sequence (Fig. 5A). Because the expression of T21D12.3 was rather confined to intestinal cells (Fig. 3, 4) and because intestinal cells play critical roles in nutritional state of nematode [16], we investigated intestinal lipid storage of these mutants in this study. To take advantage of lucent body of C. elegans, lipid storage was investigated by culturing on Nile Red-including dishes. It is known that the in vivo staining with Nile Red does not affect PLoS ONE | www.plosone.org 2 January 2009 | Volume 4 | Issue 1 | e4104