(A) Genomic region of Ubx gene, exons are illustrated in boxes (red: non-coding regions, blue: coding regions) and introns in lines, orange box highlights the 1st exon. (B) close up of the 1st exon targeted by guide RNAs (green arrows) for CRISPR/Cas9, below: construct used for homologous recombination, recombination event illustrated by the crossed lines. Red: non-coding region, blue: coding region, green: GFP coding region, yellow: spacer containing a TEV cleavage site with TIPI-degron (TEV protease- induced protein inactivation) (C, D) Lateral view of stage 14 GFP-Ubx Drosophila embryos stained for the muscle differentiation marker Mef2 (blue) (C), the pan-neuronal marker Elav (blue) (D), Ubx (red) and GFP green). Boxes indicate the location of the close-ups on the right panel. (E, F) Lateral view of stage 15 arm-GAL4,GFP-Ubx and arm >Nslmb,GFP-Ubx Drosophila embryos stained for GFP (green) to highlight Ubx expression. The GFP staining is mostly lost in the ectoderm, mesoderm and neuronal system when Ubx is degraded (F) as compared to the control (E). (G) Quantification of GFP signal intensity in the whole arm-GAL4,GFP-Ubx and arm >Nslmb,GFP-Ubx Drosophila embryos, showing that GFP is strongly decreased (***=p < 0.001). (H–K) Lateral view of first instar larva cuticles, heterozygous (H) and homozygous Ubx mutants (I), homozygous GFP-Ubx (J), heterozygous Ubx mutant over GFP-Ubx (K), asterisk indicates the 1st abdominal segment (A1), homozygous Ubx mutants show a loss of the denticle belts in A1 compared to the heterozygous control and the GFP-Ubx line, the Ubx mutant phenotype can be rescued with the GFP-Ubx. (L) Quantification of the rescue experiment, showing that no Ubx phenotype is detachable in the Ubx1/TM6 with GFP-Ubx cross, indicating that GFP-Ubx is fully functional during embryonic and larval development. (M, N) Lateral view of first instar larva cuticles, arm-GAL4,GFP-Ubx (M) and arm >Nslmb,GFP-Ubx (N), asterisk indicates A1, arm >Nslmb,GFP-Ubx larvae (N) shows a loss of the A1 denticle similar to the homozygous Ubx mutant (I) when compared to arm-GAL4,GFP-Ubx (M). (O) GFP protein immunoprecipitation (IP) experiments in Drosophila embryos, comparison of GFP-nanobody (deGrad) and TEV-TIPI protein degradation efficiency, control lines: arm-GAL4,GFP-Ubx (arm-GAL4), UAS-Nslmb,GFP-Ubx (UAS-Nslmb), UAS-TEV-P14;GFP-Ubx (UAS-TEV-P14), crosses: arm-GAL4,GFP-Ubx x UAS-Nslmb,GFP-Ubx (arm >Nslmb), arm-GAL4,GFP-Ubx x UAS-TEV-P14;GFP-Ubx (arm >TEV P14), expected size of GFP-Ubx 80 kDa, IPs are probed with antibodies indicated on the right, lane 1–5 are 2.5% input of each sample, lane 6–10 GFP-trap bead IP-experiments for each sample, control samples indicate bands for GFP-Ubx using GFP or Ubx antibodies, arm >Nslmb shows no band for GFP-Ubx, whereas arm >TEV P14 displays the same band as the control samples, indicating that the degradation of the GFP-Ubx with GFP-nanobody technology is more efficient than with the TEV-TIPI system. (P) Upper panel: Venn diagram representing the overlap between the neuronal stage 10–13 transcriptome (purple) and the genes up-regulated in mesodermal nuclei after Ubx depletion (RNAup UbxDeGrad) (pink). Lower panel: Fold enrichment of gene ontology terms of the 578 genes expressed in neuronal cells and up-regulated in mesodermal cells after Ubx depletion.