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. 2009 Jan 22;583(2):456-64.
doi: 10.1016/j.febslet.2008.12.046. Epub 2008 Dec 29.

Characterization of the Caenorhabditis elegans Islet LIM-homeodomain ortholog, lim-7

Roumen Voutev  1 , Ryan Keating, E Jane Albert Hubbard, Laura G Vallier
Affiliations

Characterization of the Caenorhabditis elegans Islet LIM-homeodomain ortholog, lim-7

Roumen Voutev et al. FEBS Lett. .

Abstract

lim-7 is one of seven Caenorhabditis elegans LIM-homeodomain (LIM-HD)-encoding genes and the sole Islet ortholog. LIM-HD transcription factors, including Islets, function in neuronal and non-neuronal development across diverse phyla. Our results show that a lim-7 deletion allele causes early larval lethality with terminal phenotypes including uncoordination, detached pharynx, constipation and morphological defects. A lim-7(+) transgene rescues lethality but not adult sterility. A lim-7(+) reporter in the full genomic context is expressed in all gonadal sheath cells, URA neurons, and additional cells in the pharyngeal region. Finally, we identify a 45-bp regulatory element in the first intron that is necessary and sufficient for lim-7 gonadal sheath expression.

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Figures

Figure 1
Figure 1. The lim-7 locus and reporter constructs, LIM-7 structure, and gonadal sheath expression of the Intron 1 element
(A) Upper, lim-7 genomic structure. Black and white exons correspond to domains of LIM-homeodomain proteins depicted in (B): black, LIM-domains; and white, homeodomain; dark gray, non-domain sequences. Lower, intron 1 fusions to GFP. (+), expression in the gonadal sheath; (−), no expression in the gonadal sheath; numbering refers to nucleotides from the beginning of intron 1. (B) LIM-7 LIM-HD protein structure and predicted tm674 mutant protein structure. Color scheme as in (A). (C) Expression of pGC433 in the gonadal sheath. Gonad is outlined and maturing oocytes are outlined by dashed lines. Arrows indicate the nuclei of the thin gonadal sheath cells in plane of focus; arrowheads indicate germ cell nuclei surrounded by gonadal sheath (fluorescence comes from sheath); black asterisk marks the proximal end of the gonad, where sheath pair 5 is located (fluorescence to left of asterisk may be due to lim-7(+)::mCherry fluorescence in Sh5; white asterisks in merge depicts the location of the three sheath nuclei in focus. (D) Expression of pGC433 in the head. Note neuron with anteriorly directed process encircling pharyngeal isthmus in upper animal (depicted in black in cartoon to right of images). (C) and (D): Left panel, DIC; middle panel, GFP fluorescence; right panel, merged image. Scale bar, 20 μm.
Figure 2
Figure 2. Terminal phenotypes of tm674.
Major phenotypes of lim-7(tm674) include Unc, Pun, Dpy, constipation, defective head morphology, and increased body vacuolarization. All images are of L1 larvae visualized using DIC optics. (A) Unc, Pun and constipation phenotypes. (B) Head bump, normal pharyngeal attachment, and increased body vacuoles. (C) Dpy, Pun and constipation. (D) Rare gross head malformation. Inset is magnification of head for better detail. Arrowheads in A, B, C, end of pharynx; Arrows in A, C, distended intestine; * in B, body vacuoles. Scale bars, 20 μm.
Figure 3
Figure 3. Expression pattern of a lim-7(+)::mCherry fosmid-based reporter
RW10289 animals were examined for mCherry fluorescence. Because the gonadal sheath is a very thin tissue that surrounds the proximal two-thirds of the gonad, sheath nuclei are difficult to discern without a fluorescent marker. (A) Adult gonad showing six sheath cell nuclei; additional expressing sheath nuclei out of focal plane. (B) Adult gonad showing sheath pair five expressing lim-7(+)::mCherry plus two other sheath nuclei. Arrowheads mark sheath pair five nuclei. In A and B cartoons, sheath nuclei depicted by white asterisks; pale gray structure, spermatheca; large white ovals, fertilized embryos; maturing oocytes outlined by dashed lines. (C) and (D) L1 larvae expressing lim-7(+)::mCherry in URA neurons and additional cells near the pharyngeal isthmus. (E) Adult pharyngeal expression. (F) Late embryo pharyngeal isthmus expression. Drawing includes only pharyngeal bulbs and isthmus of the embryo; other regions of embryo not drawn. (C–F) Asterisks in drawings represent main fluorescent nuclei location in pharyngeal region. Left, DIC; middle, lim-7(+)::mCherry expression; right, merged image; far right, cartoon of relevant structures in each panel. (C – F), asterisks represent fluorescent nuclei. Scale bars, 20 μm.
Figure 4
Figure 4. tm674 hermaphrodites bearing a lim-7(+) transgene exhibit gonadal defects resulting in sterility
(A) Adult gonad from tm674; lvEx4[lim-7(+); sur-5::GFP] (strain HN15, see Materials and Methods section 2.1). Distal gonad marked by (*), single disintegrating embryo in the uterus (white arrow), and loose sperm, (white arrowhead). (B) RW10289 gonad for comparison. Cartoon of each gonad (in dark gray) is to the right: Maturing oocytes outlined by dashed lines and immature germ cell nuclei indicated by open circles. The proximal gonad is adjacent to the spermatheca, in pale gray; embryos are open ovals to the left of spermatheca; black filled circles in (A) are loose sperm. Scale bar, 20 μm.
Figure 5
Figure 5. Intron 1 sequence elements from lim-7 orthologs in four Caenorhabditis species
(A) The 125-bp region in intron 1 necessary for gonadal sheath expression; bold underlined nucleotides (690 – 735) are necessary and sufficient for expression in the gonadal sheath. (B) The highlighted 21-nt element conserved in all four species. (C) The highlighted 22-nt element conserved in three of the four species. All numbering begins from first nucleotide of the 5′ end of intron 1.
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