Picture from Wittes J et al. (2024) MicroPubl Biol "New Flexon-based reagents for tissue-specific Auxin-Inducible Degradation ...."

Figure 1. Flexon-based reagents for tissue-specific expression of TIR1 and for characterizing Cre and Flp recombinase excision patterns: A) A Flexon stop cassette is an artificial exon that contains stop codons and a frameshift generator (***), leading to termination of protein translation and nonsense-mediated decay of the transcript. Triangles represent recombinase target sites and the thin line represents intronic sequences flanking the artificial exon. For further information and considerations about Flexon design and placement see Shaffer and Greenwald (2022). B) TIR1(flexon) is expressed under the control of a strong ubiquitous promoter from rps-27 (rps-27p). In the absence of Cre, the Flexon cassette interrupts TIR1 translation after codon 159. In the presence of Cre, the Flexon is excised and the complete TIR1 protein is expressed under the control of rps-27p. TIR1F79G(flexon) has an identical design to TIR1(flexon). C) The design of Cre and Flp testers was based on the design of arTi361[rps-27p::GFP(flexon)::H2B::unc-54 3'UTR] (Shaffer and Greenwald 2022). Nuclearly-localized GFP is expressed under the control of rps-27p after excision in the presence of an appropriate Cre or Flp driver. H2B represents the sequence of a histone gene, his-58. All transgenes reported in this study use the neutral unc-54 3'UTR.

Picture from Wilkinson HA et al. (1994) Cell "Reciprocal changes in expression of the receptor lin-12 and its ligand lag-2 ...."

Figure 3. Expression of lin-12::lacZ and lag-2::lacZ in Z1.ppp and Z4.aaa in lin-12(+) Hermaphrodites. Open arrow indicates Z1.ppp; solid arrow indicates Z4.aaa. Preforming, forming, and formed refer to the morphology of the gonad primordium (see description in text): preforming, Ll molt/early L2 stage; forming, mid-late L2 stage; formed, L2 molt. Anterior is to the left. All preforming and forming primordia are shown from a ventral view; in forming primordia, the identities of the nonstaining cells is inferred by their position relative to other somatic gonadal cells and to the anterior-posterior and left-right axes. Formed primordia are shown from a dorsal view; in the primordia shown, Zi .ppp has become the AC. The lin-12:lacZ reporter gene is also expressed in Z1.ppa and Z4.aap. Here, in the pictures of the forming stage, expression in Z1.ppa is visible in this plane of focus, and in the pictures of the preforming and formed stages, expression in Z4.aap is visible. lin-12 activity influences, but is not absolutely required to specify, the fates of Z1.ppa and Z4.aap in the L2 stage (Greenwald et al., 1983; Seydoux et al., 1990). Other cells stain in other planes of focus (Wilkinson, 1994), and can sometimes be seen as dark areas in the photograph. A complete description of the spatial and temporal pattern of lin-12 expression in hermaphrodites will be described elsewhere (Wilkinson, 1994; H. A. W. and I. G., unpublished data).Complete genotypes: uric-54 smg-1; arIs11 [lin-12::lacZ] or unc-54 smg-1; arIs13[lag-2::lacZ].

Picture from Karp X et al. (2003) Genes Dev "Post-transcriptional regulation of the E/Daughterless ortholog HLH-2, ...."

Figure 2. Dynamic pattern of HLH-2 protein accumulation during the AC/VU decision. (A-F) Confocal micrographs of L2 hermaphrodite gonads. Hermaphrodites stained with antibodies against HLH-2 (green) and LacZ (red); cells in which both antibodies are detected appear yellow. Small yellow arrows indicate Z1.ppp and Z4.aaa. Larger white arrows indicate HLH-2 expression in the presumptive AC. Distal tip cells are marked with an asterisk. Photomicrographs are arranged in an inferred temporal sequence, on the basis of the observation that lin-12::lacZ and lag-2::lacZ are initially expressed in both Z1.ppp and Z4.aaa, but become restricted to the presumptive AC (lag-2::lacZ expressing) or presumptive VU (lin-12::lacZ expressing). Only (and all) animals that showed HLH-2 expression in both distal tip cells as well as lin-12::lacZ expression in all six vulval precursor cells (Wilkinson and Greenwald 1995) or lag-2::lacZ expression in both distal tip cells (Henderson et al. 1994) were counted, to be certain that the staining procedure had worked well in each animal scored. (A,C,E) lin-12::lacZ animals (full genotype: smg-1(r861) unc-54(r293); arIs11[lin-12::lacZ]). A total of 36 animals were observed with lin-12::lacZ in both Z1.ppp and Z4.aaa. Of these, 20/36 animals showed no HLH-2 accumulation in either cell (A), and 16/36 showed HLH-2 accumulation in one cell (C). Seven animals were observed in which lin-12::lacZ expression was already restricted to the presumptive VU. Of these, 7/7 showed HLH-2 accumulation in the other cell, the presumptive AC (E). (B,D,F) lag-2::lacZ animals (full genotype: smg-1(r861) unc-54(r293); arIs13[lag-2::lacZ]). Seven animals were observed with lag-2::lacZ expressed in both Z1.ppp and Z4.aaa. Of these, 2/7 animals showed no HLH-2 accumulation in either cell (B), and 5/7 showed HLH-2 accumulation in one cell (D). A total of 21 animals were observed in which lag-2::lacZ expression was restricted to the presumptive AC. Of these, 21/21 showed HLH-2 accumulation costaining the presumptive AC (F).

WBPicture0000015178

Figure 1. daf-16 isoform-specific mutants express VPC specification markers during dauer:(A) Vulval precursor cell (VPC) specification occurs in the L3 stage during continuous (non-dauer) development (Sternberg 2005). Six equipotential VPCs are named P3.p-P8.p. LIN-3/EGF is secreted from the anchor cell (AC) and a canonical EGFR/Ras/MAPK pathway specifies 1˚ vulval fate in P6.p (red). P6.p then expresses ligands for LIN-12/Notch, activating LIN-12 signaling in the adjacent cells, P5.p and P7.p. LIN-12 signaling specifies 2˚ vulval fate (blue). The remaining VPCs adopt a non-vulval fate (Sternberg 2005). (B) As larvae molt from L2d into dauer (L2d-D molt), 1˚ cell fate marker expression is observed in P6.p (red). However, this expression is lost within one day of entering dauer, such that larvae that have been in the dauer stage for one day ("1-day dauer") do not express 1˚ fate markers. 2˚ cell fate markers are not observed prior to dauer or during dauer in wild-type or daf-7 control larvae (Karp and Greenwald 2013). (C-D) The daf-7(e1372) allele was used to induce dauer formation and is present in all strains. Strains lacking one or more daf-16 isoform were scored for the presence or absence of expression of a 1˚ fate marker (arIs131[lag-2p::yfp]) or 2˚ fate marker (arIs116[lst-5p::yfp]). Individual dots represent the percent of larvae expressing the relevant marker in a single trial of 5-36 dauer larvae. The mean and standard deviation for each strain is shown. Letters above the data indicate statistical groupings, where strains that have a letter in common are not statistically different from each other. (Kruskal Wallis with Dunn's multiple comparison test, α = 0.05). P-values for all non-significant comparisons were > 0.99 with the following exceptions: In panel (C), daf-16(0) vs. daf-16(a), p = 0.08. In panel (D), control vs. daf-16(a1), p = 0.19. Control vs. daf-16(a,f), p = 0.19. daf-16(0) vs. daf-16(f), p = 0.08. (C) Percent of 1-day dauer larvae that express the lag-2p::yfp 1˚ fate marker in P6.p. daf-16 alleles: daf-16(0), mgDf50. daf-16(a), tm5030. daf-16(f), tm6659. daf-16(a,f), mg54. Loss of daf-16a and daf-16f together recapitulates the phenotype of loss of all daf-16 isoforms in the null allele daf-16(0). (D) Percent of early dauer larvae that express the lst-5p::yfp 2˚ fate marker in P5.p and/or P7.p. daf-16 alleles: daf-16(0), mgDf50. daf-16(a1), tm5030. daf-16(a2), tm5032. daf-16(f), tm6659. daf-16(a,f), mg54. Loss of each daf-16 isoform tested produces a phenotype intermediate between the control daf-7 strain and the daf-16(0) allele.