Figures and data
ifc regulates CNS and glial morphology.
A) Ventral views of late-third instar larvae of indicated genotype showing 3xP3 RFP labeling of CNS and nerves. Arrowheads indicate nerve bulges; scale bar is 200μm. B-B’) Schematic of Ifc (B) and human DEGS1 (B’) proteins indicating location and nature of ifc mutations and 15 HLD-18-causing DEGS1 mutations.7,8,9 C) Schematic of de novo ceramide biosynthesis pathway indicating the subcellular location of ceramide synthesis and ceramide modifications. D) Chemical structure of dihydroceramide and ceramide; arrow indicates trans carbon-carbon double bond between C4 and C5 in the sphingoid backbone created by the enzymatic action of Ifc/DEGS1. E) Normalized quantification of the relative levels of dihydroceramide, ceramide, and six related sphingolipid species in the dissected CNS of wild-type and ifc-/- late-third instar larvae. F) Ventral views of Drosophila CNS and peripheral nerves in wild-type and ifc-/- mutant late-third instar larvae labeled for NCAD to mark the neuropil, HRP to label axons, RFP to label glia, Dpn to label neuroblasts, ELAV to label neurons, and FABP to label cortex glia. Anterior is up; scale bar is 100μm for whole CNS images and 20μm for peripheral nerve image. Statistics: * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001, **** denotes p < 0.0001.
Loss of ifc disrupts glial morphology.
A-E’) High magnification ventral views and X-Z and Y-Z projections of the nerve cord of wildtype and ifc-/- late third instar larvae labeled for ELAV (magenta) for neurons and Myr-GFP (green) for cell membranes of indicated glial subtype. Anterior is up; scale bar is 40μm. F-J’) High magnification views of individual glial cells of indicated glial subtype in the nerve cord of wildtype and ifc-/- larvae created by the MultiColor-FlpOut method.33 Anterior is up; scale bar is 20μm. K-N) Quantification of total number of indicated glial subtype in the nerve cord of wildtype and ifc-/- late third instar larvae (n = 7 for K, L, N; n = 6 for M). Statistics: * denotes p < 0.05, **** denotes p < 0.0001, and ns, not significant.
ifc acts in glia to regulate CNS structure and glial morphology.
A-I) Ventral views of photomontages of the CNS of late third instar larvae labeled for FABP (greyscale) to mark cortex glia in late third instar larvae of indicated genotype. Neuronal-specific transgene expression was achieved by using elav-GAL4 combined with repo-GAL80; glial-specific transgene expression was achieved by using repo-GAL4. J-K) Quantification of the number of swollen cortex glia in the abdominal segments of the CNS of late-third instar larvae of the indicated genotype for the RNAi (J) and gene rescue assays (K). Statistics: * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001, **** denotes p < 0.0001, and ns, not significant.
Loss of ifc drives ER expansion in cortex glia.
A-E) Dorsal (A, B-B’’, and C-C’’) and ventral (D-D’’ and E-E’’) views of the CNS of late-third instar wild-type larvae labeled for ifc RNA (grey in A; magenta in B’), ifc-GAL4>nRFP (magenta; C’-E’), EBONY to mark astrocytes (green; B and C), REPO to mark glia (green; D), and ELAV to mark neurons (green; E). Panels D-D’’ and E-E’’ show surface and interior views, respectively, along the Z-axis on the ventral side of the nerve cord. Arrowheads in E-E’’ identify neurons with low level ifc-GAL4 expression. F-G) High magnification ventral views of thoracic segments in the CNS of wild-type late third instar larvae labeled for GFP (green; F and G), CNX99A (magenta; F’), ESYT (magenta; G’). H-M) Late third instar larvae of indicated genotype labeled for 3xP3-RFP (green; H’-M’), CNX99A (magenta; H and K), GOLGIN84 (magenta; I and L), and LAMP (magenta; J and M). Anterior is up; scale bar is 100μm for panel A and 30μm for panels B-M.
Loss of ifc leads to internal membrane accumulation and lipid droplet loss in cortex glia.
A-E’) TEM images of cortex glia cell body (A-A’ and B-B’) and neuronal cell bodies (C-C’ and D-D’) at low (A-A’) and high (B-B’, C-C’, and D-D’) magnification in the nerve cord of wildtype (A-D) and ifc-/- (A’-D’ and E-E’) late third instar larvae. (A-A’) Dotted lines demarcate cell boundary of cortex glia; yellow squares highlight regions magnified in B, B’, and E’. Scale bar is 3μm for A and A’ and 1μm for B-B’. (B-B’) Cy denotes cytoplasm; Nu denotes nucleus. Solid white arrows highlight the layered internal membranes that occupy the cytoplasm of ifc-/- cortex glia. (C-C’ and D-D’) Black arrows highlight cortex glia membrane extensions that enwrap neuronal cell bodies; hollow white arrows denote the absence of cortex glia membrane extensions; white asterisk denotes lipid droplets. Scale bar is 2μm. E-E’) An additional example of membrane-filled cortex glia cell body in ifc-/- larvae. Scale bar is 2μm for E and 1μm for E’. F) Cortex glia in ifc mutant larvae labeled for Myr-GFP (green) to label membranes and CNX99A to label ER membranes. Scale bar is 30μm. G-H) Black and white and colored TEM cross-sections of peripheral nerves in wild type and ifc-/- late-third instar larvae. Blue marks perineurial glia; purple marks subperineurial glia; pink marks wrapping glia. Scale bar: 2μm. I-I’) High magnification ventral views of abdominal segments in the ventral nerve cord of wild-type (I) and ifc mutant (I’) third instar larvae labeled for BODIPY (green) to mark lipid droplets and FABP (magenta) to label cortex glia. Anterior is up; scale bar is 30μm. J) Graph of log-fold change of transcription of five genes that promote membrane lipid synthesis in ifc-/- larvae relative to wildtype. Dotted line indicates a log2 fold change of 0.5 in the treatment group compared to the control group. -M) Quantification of the number (G) and area of lipid droplets (H and I) in the dissected CNS of wildtype and ifc-/- larvae. Statistics: * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001, **** denotes p < 0.0001, and ns, not significant.
PC, PE, PS, and TG exhibit higher saturation levels in a CNS-specific manner in ifc mutant late third instar larvae.
A-C) Quantification of total (A) and species-specific (B and C) TGs in whole larvae (A and C) and dissected CNS (A, B, and C) of wildtype and ifc-/-larvae. D-F). Quantification of total (D) and species-specific (E and F) PCs in whole larvae (D and F) and dissected CNS (D, E, and F) of wildtype and ifc-/-larvae. G-I) Quantification of total (G) and species-specific (H and I) PEs in whole larvae (G and H) and dissected CNS (G, H, and I) of wild-type and ifc-/- larvae. J-L) Quantification of total (J) and species-specific (K and L) PSs in the whole larvae (J and L) and dissected CNS (J, K, and L) of wild-type and ifc-/- larvae. Statistics: * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001, **** denotes p < 0.0001, and ns, not significant.
Glial-specific knockdown of ifc triggers neuronal cell death.
A-D) TEM images of cortex glia cell body (A and B) and neuronal cell bodies (C and D) at low (A) and high (B, C, and D) magnifications in the nerve cord of ifc-/-; repo>schlank RNAi late third instar larvae. Dotted lines demarcate cell boundary of cortex glia; yellow squares highlight regions magnified in A. Scale bar: 3μm for A, 1μm for B, and 2μm for C and D. (B) Cy denotes cytoplasm; Nu denotes nucleus. (C-D) Black asterisk denotes lipid droplets. E-F) Ventral views of abdominal sections of CNS of ifc-/-; UAS-schlank RNAi/+ larvae (E) and ifc-/-; repoGAL4/UAS-schlank RNAi larvae (F) labeled for neurons (ELAV, Green) and cortex glia (FABP, magenta/grey). Scale bar is 30μm for E-F. G-L) Low (G-L) and high (G’-L’ and G’’-L’’) magnification views of the brain (G-I) and nerve cord (J-L) of late-third instar larvae of the indicated genotypes labeled for ELAV (magenta or greyscale) and Caspase-3 (green). Arrows indicate regions of high Caspase-3 signal and/or apparent neuronal cell death identified by perforations in the neuronal cell layer. Scale bar is 50μm for panels G-L and 10μm for panels G’-L’’. M-N) Quantification of CNS elongation (M) and 3xP3 RFP intensity (N) in ifc mutants alone, ifc mutants with one copy of schlank[G0365] loss-of-function allele, or ifc mutants in which schlank function is reduced via RNAi in glial cells. O) Quantification of the area of lipid droplets in dissected CNS of ifc mutants and ifc mutants in which schlank function is reduced via RNAi in glial cells. Anterior is up in all panels. P-Q) Quantification of Cleaved Caspase-3 neurons for panels G-I (P) and J-L (Q). Statistics: * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001, **** denotes p < 0.0001, and ns, not significant.
Ceramide metabolic changes in whole larvae.
A) Normalized quantification of the relative levels of dihydroceramide, ceramide, and six related sphingolipid species in the whole larvae of wild-type and ifc-/- late-third instar larvae. B) Chemical structure of dihydroceramide phosphoethanolamine (DiCPE), ceramide phosphoethanolamine (CPE), glucosyl-dihydroceramide (Glc-DiCer), glucosyl-ceramide (GlcCer), sphinganine, and sphingosine; arrow indicates the cis carbon-carbon double bond between C4 and C5 in the sphingoid backbone created by the enzymatic action of Ifc/DEGS1.
The M{3xP3-RFP.attP}ZH-51D transgene insert labels most glia.
Low and high magnification images of late third instar larvae that harbor the M{3xP3-RFP.attP}ZH-51D transgene showing RFP expression and markers for different glial subtypes. A) Cortex glia: Ventral view of full CNS labeled for FABP to label cortex glia and RFP. Inset shows high magnification view of the brain with clear overlap of FABP and RFP expression (arrows).
B) Astrocyte-like glia: Dorsal view of thoracic and abdominal region of CNS labeled for Ebony to label astrocyte glia and RFP; arrows highlight overlap of RFP and Ebony expression in some astrocyte-like glia. C) Ensheathing glia: Dorsal view of thoracic and abdominal region of CNS labeled for RFP and Myr-GFP. The UAS-Myr-GFP transgene is driven by the ensheathing glia-specific GAL4 driver line GMR56F03. Note the colocalization of RFP and GFP in the nerves exiting the CNS and the circular structures of ensheathing glia near the dorsal midline. D) Subperineurial glia: The two left-most panels show ventral views of thoracic and abdominal region of CNS labeled for RFP and Myr-GFP driven by the GMR54C07-GAL4 line, which is specific to subperineurial glia. Arrows identify nuclei of subperineurial glia, which contain RFP and are encircled by Myr-GFP. Right panel shows cross section of CNS that highlights RFP and Myr-GFP co-expression in subperineurial glia at lateral edge of CNS (arrows). E) Perineurial glia: Ventral view of brain labeled for RFP and Myr-GFP driven by the GMR85G01-GAL4 line, which is thought to be specific for perineurial glia. Arrows point to the small GFP-expressing perineurial glia, most of which do not express detectable levels of RFP. Visual inspection and quantification of the relative expression of RFP to each glial subtype marker in at least five brain-nerve cord complexes revealed that essentially all cortex, astrocyte, ensheathing, and subperineurial glia expressed RFP, but that astrocyte-like glia expressed RFP at much lower levels than the other three glial subtypes, and that most perineurial glia did not express RFP.
Most 3xP3-GFP or 3xP3-RFP transgenes label glia.
Low magnification views of the indicated 3xP3 transgenes driving GFP (left) or RFP (right) in the CNS. Low magnification views illustrate that the three 3xP3 transgenes label or highlight the CNS. High magnification views illustrate that all three transgenes label cortex glia (arrowheads), which are marked by FABP. Two of the three transgenes strongly label astrocyte-like glia (arrowheads), which are marked by Ebony. Two of the three transgenes also clearly label ensheathing glia (arrows). Scale bar is 100 microns for low magnification images; 50 microns for high magnification images. We note that all M{3xP3-RFP.attp} transgenes (n=4), all Mi{GFP[E.3xP3]=ET1} transgenes (n=3), and all Tl{GFP[3xP3.cLa]=CRIMIC.TG4} transgenes (n=6) that we assayed labeled the CNS in a manner similar to that detailed here for the indicated lines. We note that we saw no GFP CNS expression in the TI{GFP[3xP3.cLa]=KozakGAL4}spag4[CR70688-KO-kG4]/SM6a line. All lines that we tested are listed below:
Mi{GFP[E.3xP3]=ET1} transgenes tested: y[1] w[67c23]; Mi{GFP[E.3xP3]=ET1}MB00051: BDSC-22685 y[1] w[67c23]; Mi{GFP[E.3xP3]=ET1}MB00056: BDSC-22686 w[1118]; Mi{GFP[E.3xP3]=ET1}Ppn[MB07666]: BDSC 25277
Tl{GFP[3xP3.cLa]=CRIMIC.TG4} transgenes tested:y[1] w[*]; TI{GFP[3xP3.cLa]=CRIMIC.TG4.0}mfr[CR70821-TG4.0]: BDSC 98524: y[1] w[*]; TI{GFP[3xP3.cLa]=CRIMIC.TG4.0}lace[CR70094-TG4.0]/SM6: BDSC 92702 y[1] w[*] TI{GFP[3xP3.cLa]=CRIMIC.TG4.2}schlank[CR01622-TG4.2] Josd[CR01622-TG4.2-X]/FM7: BDSC 91266 y[1] w[*]; TI{GFP[3xP3.cLa]=CRIMIC.TG4.0}bwa[CR02807-TG4.0]/SM6a; BDSC 97620 y[1] w[*]; TI{GFP[3xP3.cLa]=CRIMIC.TG4.2}ifc[CR70115-TG4.2]/SM6a; BDSC 92710 y[1] w[*]; TI{GFP[3xP3.cLa]=CRIMIC.TG4.1}ghi[CR01805-TG4.1]/TM3; BDSC 92656
M{3xP3-RFP.attp} transgenes tested M{3xP3-RFP.attP}ZH-2A: Derived from BDSC 24480 M{3xP3-RFP.attP’}ZH-22A: Derived from BDSC 24481 M{3xP3-RFP.attP’}ZH-51C: Derived from BDSC 24482 {3xP3-RFP.attP}ZH-51D: Derived from BDSC 24483
Newly generated FABP antibody labels cortex glia.
Ventral views of the brain and ventral nerve cord of wild-type larvae that express Myr-GFP under the control of cortex-specific GAL4 driver line GMR54H02. Myr-GFP (green) highlights the cell membranes of cortex glia; FABP protein expression (red) exhibits significant overlap with Myr-GFP. Anterior is up; scale bar is 50 microns.
Loss of ifc function reduces optic lobe size and optic lobe neuroblast number.
Ventral view of single brain hemisphere of late-third instar larvae of the indicated genotype labeled for neuroblasts (DPN; green), glia (RFP, red), and neurons (ELAV, blue). Loss of function in ifc results in reduction of the number of optic lobe neuroblasts (brackets) and the size of the optic lobe (dotted circles) relative to wild-type and ifc mutants in which schlank function is inhibited specifically in glia (repo-GAL4>UAS-schlankRNAi). Anterior is up; scale bar is 50 microns.
Loss of ifc increases RFP fluorescence and induces the formation of bright RFP-positive puncta or aggregates.
A-B) Ventral views of the CNS of wildtype (A) and ifc mutant (B) late third instar larvae homozygous for the M{3xP3-RFP.attP} transgene, which drives RFP expression in most glia. RFP expression is shown in greyscale. Larvae were fixed at the same time in the same tube, mounted on the same slide, and imaged at the same time using identical parameters. ifc mutant larvae consistently exhibit higher levels of RFP expression. Anterior is up; scale bar is 100 microns. C-F) High magnification ventral views of the abdominal region of the ventral nerve cord (C and D) and brain (E and F) of wildtype (C and E) and ifc mutant larvae (D and F) showing RFP expression in greyscale. Note that the gain was increased in wildtype larvae (C and E) relative to ifc mutant larvae (D and F) to enable visualization of RFP signal in the wildtype CNS. Brightly fluorescent puncta or aggregates (arrows) appear in cortex glia of ifc mutant, but not wild-type larvae. Anterior is up. Scale bar is 50 microns for C-F. G) Quantification of relative RFP intensity between wild type and ifc mutant larvae – p<2.0×10-4.
ifcjs1 and ifcjs2 alleles drive cortex glia swelling, ER expansion in cortex glia, and neuronal cell death.
A-C) Abdominal segments of dissected ventral nerve cords from late-third instar larvae of the indicated genotype labeled for FABP to label cortex glia and ELAV to label neurons (A-A’’) or FABP and the ER-marker Calnexin-99A (CNX99A; B-B’’). As observed in ifcjs3/ifc-KO larvae, ifcjs1 and ifcjs2 larvae exhibited swollen cortex glia (A-B) and an expanded ER phenotype in cortex glia as indicated by elevated CNX99A expression (B). (C-C’’’) ifcjs1 and ifcjs1/ifcjs2larvae also exhibit enhanced presence of the neuronal cell death marker cleaved Caspase 3 (CC3). Note that the chromosome that harbors the ifcjs2 chromosome also harbors a mutation in the pro-apoptotic gene, Dark. Therefore, to track the impact of ifcjs2on neuronal cell death, we assessed neuronal cell death in larvae trans-heterozygous for ifcjs1 and ifcjs2 (Civ). Scale bar is 50 microns.
Reduction of cortex glia number in ifc-/- larvae results from increased apoptosis and reduced cell proliferation.
A-B) Thoracic regions of the CNS of larvae of the indicated genotype labeled for ELAV (magenta) to label neurons and FABP (green) to label cortex glia. C) Number of cortex glia in the thoracic region of larvae of the indicated genotype; *** denotes p value less than 3 x 10-4. D) Number of phospho-Histone H3 positive glia in the thoracic region of larvae of the indicated genotype; ** denotes p value of less than 0.01.
Glial-specific expression of ifc completely rescues the CNS phenotype of ifc mutant larvae.
Ventral views of the CNS from ifc mutant larvae (left) and ifc mutant larvae in which a wild-type ifc transgene is driven specifically in glia under the control of repo-GAL4 (middle and right panels). Each CNS is labeled for neuroblasts in green (DPN) and neurons in red (ELAV). Note the strong rescue of the CNS elongation and reduction of optic lobe size (dotted circles) and neuroblast number (arrows) observed in ifc mutant larvae relative to those that express ifc in glia. Anterior is up; scale bar is 50 microns.
ifc is predominately expressed in glia and Ifc protein mildly localizes to the Golgi where mild expansion appears to occur in the larval CNS.
A-B) Ventral (A-A’’) and dorsal (B-B’’) views of the CNS of late-third instar wild-type larvae labeled for ifc-GAL4>nRFP (magenta; A’-B’), REPO to mark glia (green; A), and ELAV to mark neurons (green; B). C-D) High magnification ventral views of thoracic segments in the CNS of wild-type late third instar larvae labeled for GFP (green; C and D), GOLGIN84 (magenta; C’), GOLGIN245 (magenta; D’). E-F) Late third instar larvae labeled for 3xP3-RFP (green; E and F), GOLGIN245 (magenta; E’ and F’). Anterior is up; scale bar is 30μm for all panels.
ER chaperones are mostly downregulated, and genes involved the Lands cycle are mostly upregulated in the CNS of ifc-/-mutant larvae.
A) Four of the five ER chaperones in Drosophila (Schroder & Kaufman, 2006; Ryoo et al., 2007) are significantly downregulated in transcription, suggesting an atypical unfolded protein response (UPR) following the transcriptional upregulation of Xbp-1s in the ifc-deprived CNS of ifc mutant larvae. B) Schematic of the Lands cycle that functions in the acyl chain remodeling of membrane phospholipids. C-D) Most genes participating in the assembly (C) and removal (D) of acyl chains in the Lands cycle are significantly upregulated.
Glial-specific inhibition of schlank suppresses the ifc swollen cortex glia phenotype.
High magnification X-Y, X-Z, and Y-Z views of the abdominal region of the ventral nerve cord of late third instar of the indicated genotype. FABP staining (green) labels cortex glia; CNX99A staining (red) labels ER. The swollen cortex glia in ifc mutant larvae (arrows) are not apparent in either wild-type larvae or ifc mutant larvae in which schlank function was inhibited specifically in glia (ifc-/-; repo-GAL4/UAS-schlankRNAi). Anterior is up; scale bar is 20μm.
Glial-specific, but not neuronal-specific, knockdown of ifc drives neuronal cell death.
Low (A-C) and high (A’-C’’) magnification views of the ventral nerve cord of late-third instar larvae of the indicated genotype labeled for ELAV (magenta or greyscale) and Cleaved Caspase-3 (green). Arrows point to regions marked by Cleaved Caspase-3 staining and/or apparent neuronal cell death identified by circular perforations in the neuronal cell layer. Please note that the repoGAL80 transgene is present in elav>ifc RNAi larvae to block GAL4 function in glia (panel B-B’’). Anterior is up in all panels; scale bar is 50μm for panels A-C and 10μm for panels A’-C’’. Full genotypes of the larvae used in this experiment are as follows – A: UAS-ifc-RNAi/+; B: elavGAL4/+; repoGAL80/+; UAS-ifc-RNAi/+; C: repoGAL4/UAS-ifc-RNAi. D) Quantification of the number of Cleaved Caspase-3 positive neurons in the thorax region of the ventral nerve cord in control, neuronal-specific knockdown of ifc, and glial-specific knockdown of ifc L3 larvae.
Subperineurial glial cell membranes encircle dying neurons in ifc mutant larvae.
High magnification ventral views of the brain of wild-type (top) and ifc mutant (bottom) larvae labeled for Myr-GFP to mark the membranes of subperineurial glia (green, left panels), Death caspase protein-1 (DCP-1) to label dying neurons (red, left panels), and ELAV to mark neurons and the neuronal cell layer (blue, left panels). Arrows identify DCP-1 positive dying neurons, which are encircled by GFP-positive glial cell membranes in ifc mutant, but not wild-type, backgrounds. Anterior is up; scale bar is 10 microns. DCP-1 antibody obtained from Cell Signaling Technologies (Ab #9578)
