Fig 1.
obst-E mutants show “twiggy” pupal shapes.
(A) Schematic of the obst-E locus and mutations used in this study. Shaded in grey is the genomic structure around obst-E. The white arrow indicates the region deleted in obst-EDel. The gene structure and alternative splicing patterns of obst-E are magnified in the yellow shade. Black boxes are coding sequences and white boxes are untranslated regions. dsRNA is designed to target a portion of splice variant a mRNA that corresponds to the 35bp region at the 3’ end of exon 1 and the 468bp region at the 5’ end of exon 2 (red boxes). It is also expected to target exon 3 (red striped box), due to sequence similarity between exons 2 and 3. Arrowheads indicate locations of the CPTI insertions. In obst-EKO, the genomic region containing the entire coding sequences of both splice variants is replaced by an exogenous sequence. (B-G) Body shapes of larvae and pupae viewed from the dorsal sides. Anterior is to the left. Bar: 1mm. (H) Axial ratios (AR) of larvae and pupae of the indicated genotypes. The number of pupae/larvae measured for each genotype is shown in each graph. Error bars in this and all other figures are standard deviations. Asterisks with brackets indicate statistically significant difference (p<0.001). “n.s.” with a bracket, not significant difference. Asterisks/“n.s” without brackets, significant/not significant difference relative to wild-type.
Fig 2.
obst-E is required for shape change of the cuticle.
(A-D) Ventral parts of isolated larval cuticles/puparia of wild-type (A, B) and KO/Del, Act>b (C, D). For each panel, 2–3 images are spliced (borders shown by white lines). Anterior is to the left. a, abdominal segment; Wk, width of the denticle belt of kth abdominal segment; Ldk, length of the denticle belt; Lnk, length of the naked cuticle. Bars: 100 μm. (E) Averaged length/width ratios of cuticle in each denticle belt or naked region. The number of measured segments is shown in each graph. Asterisks, statistically significant difference (p<0.01, Student’s t-test); n.s., not significant. (F) Averaged contraction rates of cuticle in naked regions upon dehydration ex vivo. N = 33 naked regions for the wild-type and 40 naked regions for the mutant. p<10−6, Student’s t-test.
Fig 3.
obst-E is required for the formation of ridge structure in the larval cuticle, which resolves during puparium formation.
Three-dimensional structure of the late third instar larval cuticle of wild-type (A-C) and KO/Del, Act>b (D-F), and of the puparium of wild-type (G and H) and KO/Del, Act>b (I and J) at white prepupa (G and I) or 2 hours after white prepupa (H and J). A, D, G, H, I and J are projections of confocal images taken from the internal side. B and E are scanning electron micrographs of isolated larval cuticles taken from the internal side. A’, D’, G’, H’, I’ and J’ are optical cross-sections at the position of the dotted lines in A, D, G, H, I and J, respectively. C and F are schematic representations of A’ and D’, respectively. Anterior is to the left in A, B, D, E, G, H, I and J. External is to the left in A’, C, D’, F, G’, H’, I’ and J’. out, outer (external) surface of the cuticle; in, inner surface of the cuticle. Bars: 20 μm.
Fig 4.
Electron micrographs of the wild-type and KO/Del, act>b third instar larval cuticle.
Transmission electron micrographs of late third instar larval cuticle with the underlying epidermis of the wild-type (A-C) and of the KO/Del, Act>b mutant (D-F). Regions boxed in A and D are magnified in B, C, E and F. (G) Relative thickness of chitin lamellae. Lamella thickness was measured along the dashed white lines in A and D, and was normalized by the average lamella thickness. env, envelop; epi, epicuticle; pro, procuticle. Bars, 5 μm in A and D, 500nm in B, C, E and F.
Fig 5.
Obst-E localization and chitin-binding activity.
(A,B) Localization of GFP signals in a late third instar larva which has a copy of obst-E-a:GFP in the obst-EKO homozygous background. A is larval cuticle and epidermis of the whole body. The boxed region is magnified in B. B is a projection of confocal images taken from the internal side, and the optical cross-section at the position of the white line in B is shown in B’. Anterior is to the left in A and B, and external is to the left in B’. Bars, 500 μm in A, 20 μm in B. (C) Chitin-binding assay for the recombinant GST-His-Obst-E-a-His (1, 59kDa) and GST-His-Obst-E-b-His (2, 61kDa) proteins. GST-His-linker-His (3, 34kDa) was used as negative control. 1/400 of the input (1, 2) or 1/800 of the input (3) and 1/25 of the fraction bound to chitin beads (1, 2, 3) were loaded. in, input; b, bound. Arrowheads, bands corresponding to full-length proteins. (D) Chitin-binding assay for CBP. 1/4 of the input (in) and 3/4 of the bound fraction (b) were loaded. M, marker (kDa).
Fig 6.
Obst-E locally directs ridge formation.
(A) (Left) Preparation of larval cuticle and epidermis by cutting larvae open from the lateral sides. (Right) An example of cuticle and epidermis of late third instar larvae in which co-expression of obst-E dsRNA and GFP had been induced in mosaic manners, viewed from the internal side. Anterior is to the left. 6 images are spliced (borders shown by white dotted lines). (B-E) Structure of the larval cuticle relative to the distribution of GFP-positive cells in the underlying epidermis. The projection of confocal images taken from the internal side is shown on the left of each panel, and the optical cross-section at the position of the dotted line is shown on the right. Green bars indicate positions of GFP-positive cells in the cross-sections. Anterior is to the left in projections and external is to the left in cross-sections. Bars: 20 μm.
Fig 7.
Local disruption of the ridge structure results in local deformation of the pupal shape.
The pupal shapes (A-C) and the distribution of GFP-positive cells in the epidermis (A’-C’) are shown for seven individuals. All cells (GFP-positive and -negative) are stained by propidium iodide. Anterior is to the left. In A’, the epidermal cells in the anterior part of the body are mostly GFP-positive. In B’, the epidermal cells in the posterior part of the body are mostly GFP-positive. In C’, GFP-positive cells are sparse.
Fig 8.
Temporal expression of obst-E.
RNA expression profiles of the obst-E gene as a whole (upper) or of individual exons (lower) extracted from modENCODE developmental transcriptome data. em, embryo; L1, first instar larva; L2, second instar larva; L3, third instar larva; PS1-2, dark blue gut stage; PS3-6, light blue gut stage; PS7-9, clear gut stage; WPP, white prepupa; P, pupal stages; Ad, adult; M, male; F, female; Ecl, eclosion.
Fig 9.
Loss of obst-E function does not affect early larval cuticle morphology, and overexpression of obst-E-a does not induce change in cuticle morphology.
(A-C) First instar larval cuticle of wild-type (A), KO/Del, Act>b (B) and obst-EKO homozygote (C). (D, E) Second instar larval cuticle of wild-type (D) and KO/Del, Act>b (E). (F-H) First instar (F), second instar (G) and third instar (H) larval cuticle of flies in which obst-E-a overexpression was induced with Act-GAL4. Chitin is in yellow, and cells are in blue. Optical cross-sections at the positions indicated by white lines in (A-H) are shown in (A’-H’). Anterior is to the left in projections and external is to the left in cross-sections. Bars, 20 μm.
Fig 10.
A phylogenetic tree of insect Obst-E proteins.
The excerpt of the Obst-E group from the phylogenetic tree of Obst proteins shown in S9 Fig.
Fig 11.
The three chitin-binding domains (CBDs) are indicated by white bars. Each contains 6 cysteines (green, numbered 1 to 6), typical of CBDs of Obst proteins, which probably form intradomain disulfide bonds [5]. Amino acids conserved across the Obst-E proteins of the outgroup species and the fly Obst-E variants a and b are shaded in orange. Amino acids strictly and moderately conserved in the fly Obst-E-a and the outgroup Obst-E proteins but not in the fly Obst-E-b proteins are shaded in blue and light blue, respectively. Amino acids strictly and moderately conserved in the fly Obst-E-b and the outgroup Obst-E proteins but not in the fly Obst-E-a proteins are shaded in magenta and light pink, respectively. Predicted signal peptides are in grey. #1–4, substitutions of amino acids with distinct properties (see text for detail). The NCBI GI numbers and RefSeq Accession numbers are: Mdom peritrophin-1-like (1) (557773418, XP_005186362.1), Mdom peritrophin-1-like partial (755893484, XP_005190169.2), Dmel obstructor-E-b (24582018, NP_723116.1), Dana GF14303 isoform B (964110182, XP_014761527.1), Dpse GA10790 isoform B (969452698, XP_015035293.1), Dmoj GI11215 isoform B (968055805, XP_015020857.1), Dvir GJ18328 isoform B (968095078, XP_015025340.1), Apis probable chitinase 3 (193704528, XP_001947458.1), Mdom peritrophin-1-like (2) (557773412, XP_005186359.1), Mdom peritrophin-1-like (3) (557781096, XP_005190168.1), Mdom peritrophin-1-like (4) (755883016, XP_011293693.1), Dpse GA10790 isoform A (198475994, XP_001357226.2), Dmel obstructor-E isoform A (19920772, NP_608957.1), Dana GF14303 isoform A (194760944, XP_001962692.1), Dmoj GI11215 isoform A (195116449, XP_002002767.1), Dvir GJ18328 isoform A (195398367, XP_002057793.1), Amel LOC413679 isoform 1 (328787428, XP_397120.4), Tcas Cpap3-E (270297178, NP_001161915.1), Aaeg AAEL011897-PA (157130845, XP_001662027.1)and Agam AGAP009405-PA (158288205, XP_310082.4). Full species names are listed in Fig 10 and in S9 Fig caption.