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. 2020 Nov 23;55(4):483–499.e7. doi: 10.1016/j.devcel.2020.09.002

Figure 2.

Figure 2

Early, Direct Binding of TBX-37/38 to lsy-6 Is Necessary and Sufficient to Establish Competence for Transcription in the ASE Neurons

(A) GFP-TBX-37 ChIP-seq signal over the lsy-6 locus and flanking regions obtained from embryos staged at the peak of TBX-37/38 expression (90 min post 2-cell); two biological replicates. A peak of GFP-TBX-37 slightly downstream of lsy-6 (gray bar) was detected in all individual replicates (p value < 10−5 in each replicate). De-novo motif discovery within TBX-37/38 binding sites, retrieved a highly enriched motif that matches the binding site for TBX-38 derived from in vitro binding studies (Narasimhan et al., 2015). The peak in the lsy-6 locus overlaps two such motifs.

(B) Deletion of a 150-bp region containing the TBX-37/38 binding site (Δtbs) using CRISPR-Cas9, from the wild-type lsy-6 locus, caused fully penetrant transformation of ASEL into ASER, monitored by expression of the ASER-specific marker gcy-5prom::mCherry (n = 25 for each genotype).

(C) A yfp reporter inserted in the endogenous lsy-6 locus via CRISPR-Cas9 resulted in exclusive YFP expression in ASEL in embryos and larvae (n = 20 and n = 10 for each genotype, respectively). Deletion of the TBX-37/38 binding site (Δtbs) in this context completely abolished expression of YFP at every stage. ASEs are marked by a genome-integrated che-1prom::mCherry reporter. Representative images are shown.

(D) Embryos carrying the engineered lsy-6::yfp endogenous locus (C) and a heat-shockprom::tbx-37 transgene, were subjected to heat shock at the indicated times (dashed lines) (n ≥ 10 for each time point). Ectopic TBX-37 expression in the whole embryo at early time points induced additional lsy-6::yfp expression in ASER (∗heat-shock treatment alone caused variable expression in two cells in the tail). Beyond the 180-min time point, TBX-37 progressively lost ability to activate lsy-6::yfp expression in ASER or any other cell. Plot shows proportion and standard error of proportion (SEP). Representative images are shown for the 90- and 300-min time points. All scale bars represent 10 μm.