Table 1.
The SPC is conserved from yeast to humans.
Figure 1.
(A) spase12d4 is a 4 kb deletion (purple shaded region). (B) spase12EY10774 contains a transposon inserted in the second exon. (B) spase12C24 is a 303 bp deletion (purple shaded region). spase12 GR (A) is a 29 kb genomic construct.
Figure 2.
spase12 LOF results in a disorganized eye, loss of pigmentation, and melanotic mass formation.
(A) yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B where w+ marks control tissue and w- marks the clone. yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12d4 P[w+] (ey-flp; spase12d4) (B), yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12EY10774 P[w+] (ey-flp; spase12EY10774) (C) and yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12C24 (ey-flp; spase12C24) (D) eyes are disrupted compared to the control (A). Clonal tissue in ey-flp; spase12d4 eyes (B) appears lighter in color than spase12d4/+ which exhibits a strong P[w+] eye color (B''). spase12EY10774/+ (C''). spase12C24/+ (D''). Examples of melanotic masses in ey-flp; spase12d4 (B'), spase12EY10774 (C'), and spase12C24 (D') mosaic eyes. A single copy of spase12 GR rescues spase12d4 in yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12d4 P[w+], spase12 GR P[w+] (E). spase12rev complements spase12d4 (F). Ubiquitous expression of UAS-spase12 rescues spase12 phenotypes in w; ubi-gal4/UAS-spase12; spase12d4 P[w+]/FRT 82B spase12C24 flies (G).
Figure 3.
spase12 LOF disrupts development in multiple tissues.
yw ubx-flp/+; FRT 82B (M) P[w+ ubi-GFP]/FRT control (A), yw ubx-flp/+; FRT 82B (M) P[w+ ubi-GFP]/FRT 82B spase12C24 wings are crumpled and melanized (A'). Clones in the distal portion of yw hs-flp/+; FRT 82B (M) P[w+ ubi-GFP]/FRT 82B spase12C24 legs (B') are twisted and stunted compared to yw hs-flp/+; FRT 82B (M) P[w+ ubi-GFP]/FRT 82B control (B). yw hs-flp/+; FRT 82B (M) P[w+ ubi-GFP]/FRT 82B control (C). Clones in the head region of yw hs-flp/+; FRT 82B (M) P[w+ ubi-GFP]/FRT 82B spase12C24 result in eye, bristle, and antennal defects as well as melanotic mass formation (C').
Figure 4.
spase12 adult eyes have ectopic and missing rhabdomeres and polarity defects.
Thin plastic sections of yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B control (A), yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12d4 P[w+] (ey-flp; spase12d4) (B), yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12EY10774 P[w+] (ey-flp; spase12EY10774) (C) and yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12C24 (ey-flp; spase12C24) (D) mosaic eyes are disorganized and exhibit spacing defects between ommatidia (red asterisks) and varying rhabdomere numbers. Red circles mark ommatidia with an ectopic inner rhabdomere while yellow circles mark ommatidia with a missing inner rhabdomeres. Polarity of individual ommatidia within ey-flp; 82B (A', A''), ey-flp; spase12d4 (B', B''), ey-flp; spase12EY10774 (C', C''), and ey-flp; spase12C24 (D', D'') is represented by red arrows. Section through ey-flp; spase12C24 melanotic mass (D''') reveals degenerating tissue with large black dots consistent with dying cells (red arrows).
Figure 5.
spase12 LOF leads to increased apoptosis.
yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B (ey-flp; 82B) control (A–A'') has limited Caspase expression and normal Elav expression compared to yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12C24 (ey-flp; spase12C24) (B–B'') mosaic discs in which Caspase is upregulated and Elav expression strongly disrupted.
Figure 6.
Loss of spase12 leads to defects in cell differentiation.
yw hs-flp/+; FRT 82B (M) P[w+ ubi-GFP]/FRT 82B spase12C2448 APF eye discs stained with Armadillo (Arm) (A, B, C). GFP negatively marks clones (A', B', C'). Red dashed boxes outline representative ommatidia which are highlighted in A'', B'', C''. Support cells are color-coded according to their identity: interommatidial bristles (magenta), secondary pigment cells (cyan) and tertiary pigment cells (green) (A''', B''', C'''). The center of each ommatidium contains four cone cells, which strongly express Arm, surrounded by two primary pigment cells. spase12C24 mutant tissue (B, B' and C, C') exhibit multiple defects: ommatidia missing one or both primary pigment cells (stars), ectopic IOBs (arrowheads), ectopic primary pigment cell (asterisk), and gaps in the support cell structure that allow contact between primary pigment cells of neighboring ommatidia (red arrows). (B''') A spase12C24 mutant ommatidium is misshapen and has a cluster of three IOBs. Additionally, support cells are not properly placed and the identity of three support cells (purple) cannot be determined by their shape or placement. (C''') A spase12C24 mutant ommatidium fails to maintain the appropriate pattern of cell types at the vertices and possesses an ectopic primary pigment cell (asterisk). A secondary pigment cell (red arrow) does not fully extend to separate one ommatidium from its neighbor and one side of the ommatidium has two secondary pigment cells rather than one (white arrow).
Figure 7.
spase12 LOF results in ectopic IOBs and ommatidial fusions.
Scanning electron microscopy (SEM) of (A) yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B control and (B) yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12C24at 200X, (A', B') 1000X, and (A'', B'') 2000X. Adult ey-flp; spase12C24 eyes (B–B'') are disorganized with ectopic interommatidial bristles (red arrows) and ommatidial fusions (white arrows).