Fig 1.
A) simplified map of shaggy locus showing the major proteoforms (from FlyBase). Diamonds indicate the position of insertion sites tagged by CRISPR/Cas9 with the colour representing the tag used. B) Length of each of the major Sgg proteoforms in amino acids and an indication of proteoforms sharing the same amino acid sequence (from FlyBase). C) Amino acid sequence of the C terminal exon differentiating isoform A from isoform B. The underlined sequence is a predicted ANCHOR binding region, lowercase letters indicate predicted MATH domain interaction motifs. D) modENCODE RNAseq timecourse of relative expression levels of transcripts encoding the major Sgg-PA Sgg-PB isoforms. The X axis indicates the modENCODE samples collected with the first 12 representing 2hr intervals across embryogenesis. Dotted line indicates the end of embryonic development.
Fig 2.
Immunohistochemistry and live imaging of tagged Sgg-PA and -PB proteoforms.
All stainings are with anti-FLAG. (A, D and G) wild type embryos at stage 5–6 (blastoderm), 10–11 (extended germ band) and 16 (late embryogenesis) respectively. (B, E and H) tagged Sgg-PA at similar stages showing expression in the developing CNS (arrows in E), mature CNS and PNS (arrow and arrowhead in H). (C, F and I) tagged Sgg-PB at equivalent stages showing ubiquitous expression at the blastoderm (C) and germband extension stages (F), followed by localised expression in hindgut (arrow in I), midgut (arrowhead in I) and salivary glands (white arrow in I). (J-L) live confocal images of late stage embryos. Wild type showing gut autofluorescence (J), Sgg-PAFSVS showing prominent CNS expression (K), Sgg-PBFSVS showing mesoderm expression (L). (M) lateral view of abdominal chordotonal organs from a stage 15 Sgg-PA embryo. (N and O) lateral view of the mesoderm (N) and epidermis (O) from a stage 14 Sgg-PB embryo. P) lateral view of YFP expression in abdominal chordotonal organs of a stage 15 Sgg-PA embryo. (Q) lateral view of YFP expression the epidermis of a stage 15 Sgg-PB embryo. All embryos are lateral views with anterior to the left. Scale bar in A = 100μm (applies to A-L), Scale bar in M = 20μm (applies to M-Q).
Fig 3.
Fluorescence imaging of tagged Sgg proteoforms.
(A and C) heterozygous stage 17 female embryos carrying Sgg-PA tagged with YFP and Sgg-PB tagged with mCherry showing Sgg-PA expression in CNS (A) and PNS (C) with weaker ubiquitous expression of Sgg-PB. (B and D) stage 17 heterozygous female embryos carrying reciprocally tagged lines (Sgg-PA-mCherry, Sgg-PB-YFP) highlighting Sgg-PA in the CNS (B) and PNS (D) and Sgg-PB in the mesoderm (D). (E and F) close up view of Sgg-PA in chordotonal organs (YFP in E and mCherry in F) and punctate epidermal Sgg-PB (mCherry in E) and mesodermal Sgg-PB (YFP in F). All embryos oriented anterior to the left, dorsal to the top. Scalebar in B = 100μm (applies to A-D), Scale bar in E = 20μm (applies to E and F).
Fig 4.
RNA-seq of sgg isoform mutants.
(A) heatmaps of significant expression changes in embryos maternally and zygotically null for sggisoA and zygotically null for sggisoB with significant GO enrichments indicated. (B) String interaction map constructed from genes with significantly changed expression in sggisoB zygotic nulls showing highly connected networks of peptidases, chaperone functions and cuticle biosynthesis genes (p-values indicated corrected gene ontology enrichments for the indicated terms).
Table 1.
Significant interacting proteins identified by IPAC-MS for Sgg-PA, Sgg-PB and the SggCPTI115553 protein trap line.
Fig 5.
(A) Graph of lifespan from replicate lines of progenitor controls, sggisoA null females and males as indicated. Error bars represent standard deviation. (B) Graph of locomotor activity as measured by climbing assays with replicate lines of progenitor controls, sggisoA null females and males as indicated. Error bars represent standard deviation.