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EGL-38 Pax regulates the ovo-related gene lin-48 during Caenorhabditis elegans organ development

¹ Department of Molecular Genetics, Ohio State University, Columbus, OH 43210, USA
² Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80262, USA
³ Department of Immunology, National Jewish Medical and Research Center, Denver, CO 80206, USA

View larger version (22K): [in a new window]   Fig. 1. egl-38 and lin-48 affect the development of cells in the middle region of the C. elegans hindgut (indicated with a shaded box). (A) Wild-type pattern of hindgut cell types. (B) In egl-38 mutants, the presumptive F and U cells develop like their posterior neighbors, B and Y (Chamberlin et al., 1997). The presumptive K' cell can develop like its sibling and neighbor, K. (C) In lin-48 mutants, the presumptive U cell can develop like its neighbor and lineal homolog, B. The presumptive F cell develops abnormally (asterisk; Chamberlin et al., 1999). The presumptive K' cell can develop like its sibling and neighbor, K. Int, intestine; irv, intestinal rectal valve cells (also called vir); rep, rectal epithelial cells. Anterior is towards the left, dorsal is upwards.

View larger version (33K): [in a new window]   Fig. 2. (A) Genomic clones of lin-48. Rectangles indicate exons of lin-48, with black areas corresponding to the sequences that code for the zinc-finger domain in B. Ovals indicate the position of regulatory elements lre1 and lre2 from Fig. 5. (B) lin-48 encodes an OVO zinc-finger protein. Alignment of the C2H2 zinc-finger domain from C. elegans LIN-48, Drosophila OVO and mouse OVO1 (mOVO1). Identical amino acids are highlighted in black, conserved amino acids in gray. The cysteines and histidines predicted to coordinate zinc ions are indicated with an asterisk. The amino acids affected by lin-48 mutations are indicated. sy548 is a GAC-to-TAC transversion. sa469 is a CAT-to-CGT transition. sy234 is a CCC-to-TCC transition.

View larger version (112K): [in a new window]   Fig. 3. Expression of lin-48. (A,C,E,G) Nomarski DIC micrographs of L1 larvae. (B,D,F,H) Epi-fluorescent micrographs of the same animals. The animal in A and B bears a transgene containing pTJ1038, whereas the animals in C-H bear transgenes containing pTJ1157 or a mutant derivative (see Fig. 2A). (A,B) LIN-48 tagged with GFP is localized to cell nuclei. Nuclei of F and U are indicated. The location of K and K' is marked; the nuclei of these cells are out of the plane of focus. Expression of LIN-48::GFP transgenes is at low levels, and the F cell is expressing at a slightly lower level than the other cells in this particular male larva. (C,D) Expression pattern of lin-48::gfp in wild type. The location of the hindgut and the excretory duct cell are indicated with an arrow and an arrowhead, respectively. Expression is observed in the U, F, K and K' hindgut cells, the excretory duct cell and several cells in the head in the larva. Phasmid cell expression does not initiate until late L1 stage. (E,F) Expression pattern of lin-48::gfp transgenes that include mutations in both lre1 and lre2. These mutations disrupt expression in the hindgut cells, and excretory duct expression can be reduced. (G,H) Expression pattern of intact lin-48::gfp transgenes in an egl-38(sy294) mutant animal. The mutant animal fails to express lin-48::gfp in hindgut cells. Scale bars: in A, 20 µm for A,B; in C, 20 µm for C-H.

View larger version (25K): [in a new window]   Fig. 4. The lin-48 promoter contains two redundant elements important for lin-48 expression in hindgut cells. lin-48 sequences were tested for their ability to drive expression of GFP, and are diagrammed using the conventions of Fig. 2A. Transgenes containing the mutant sequences illustrated in Fig. 5 are indicated with X. The percentages of cells expressing GFP (black bar), expressing very low but detectable levels of GFP (gray bar) or not expressing GFP (white bar) are indicated for each construct. n, number of animals scored for expression.

View larger version (31K): [in a new window]   Fig. 5. The lin-48 promoter elements share similarity with Pax-binding sites. (A) Some representative binding sites for mammalian PAX5, and a consensus sequence (Czerny et al, 1993). (B,C) Sequence of the two elements in the lin-48 promoter, and the mutant elements used to test the function of each site in vivo. Sequences that correspond to the consensus are underlined. Nucleic acids changed in the mutant promoters are in italics.

View larger version (22K): [in a new window]   Fig. 6. Mutations in egl-38 affect the expression of lin-48 in hindgut cells. Data presented as in Fig. 4.

View larger version (34K): [in a new window]   Fig. 7. The DNA-binding domain of EGL-38 specifically binds the lin-48 lre2 site with high affinity. E. coli lysate containing EGL-38 DBD was incubated with the lre2 probe at a final dilution of 1:51,200 (top), or with the CD19 control probe at 1:6400 (bottom). Competitor sequences are described in Materials and Methods. The CD19 probe corresponds to the CD19-1 element of Fig. 5.

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