Figure 3.
Secretion of laminins via the CopII pathway, from both the mesoderm and the midgut cells themselves, is required for their correct migration and MET. (a) Laminins (green) are strongly expressed in the posterior midgut cells in sna,twi mutant embryos. The posterior midgut is demarcated by dashed red lines. (b and c) In stage 13 sar1 zygotic mutants, the ICP cells (asterisks) are found more posteriorly than in wild-type (compare c with wild-type b; the same wild-type was used as in Fig. 2 d for comparison). (d) In the wild-type stage 15 midgut epithelium, two layers of laminin are found on the basal surface, one at the endoderm/mesoderm border (d′, red arrowheads) and the second on the outer surface of the mesoderm (d′, black arrowheads). Additionally, a lower level of LanB2 at the apical surface of midgut cells can be seen (d′, by white dotted line). (e) In stage 15 sar1 mutants, large intracellular punctae of laminins can be found (e, red arrows), indicating defects in laminin secretion (n = 40 embryos at stage 15; all embryos examined had multiple intracellular punctae of LanB2; representative image shown in e). (f and g) Velocity, directional persistence, and coordination values calculated from videos of wild-type (n = 11) and sar1 (n = 6) mutant embryos. n represents the average of a minimum of 15 cells in one embryo (shown as a dot). Data are presented as mean ± SEM. **, P ≤ 0.01 by unpaired two-tailed t test (see Table S1 for raw data). (h) TwiGal4 driving Sar1DN in the mesoderm leads to a loss of LanB2 staining at the endoderm/mesoderm interface (red arrowheads). Low levels of LanB2 staining can be seen throughout the mass of rounded midgut cells (black arrowheads point to the basal side of the visceral mesoderm). (i) HkbGal4 driving Sar1DN in the endoderm leads to a loss of LanB2 staining at the endoderm/mesoderm interface (red arrowheads), while LanB2 can still be seen on the opposite side of the mesoderm (black arrowheads). (j) Plots of the average fluorescence intensity (represented as mean gray value) of LanB2 in stage 15 midgut cells measured along the apical (A) to basal (B) axis of a cell (represented in b by red line). Each n represents the average of 10 cells measured in one embryo (black dotted lines). n = 6 per condition; the mean for each condition is plotted in red. In wild-type cells there is a peak of LanB2 at the endoderm mesoderm border (P1) and on the outer surface of the mesoderm (P2). In TwiG4xSar1DN, LanB2 levels are reduced, P1 appears as a broad diffuse peak, and P2 is either absent or greatly reduced (see Fig. S5 for FWMH). In HkbG4xSar1DN, the endoderm/mesoderm peak, P1, is either absent or greatly reduced, while more diffuse levels of LanB2 can still be seen on the opposite side of the visceral mesoderm (P2 FWMH increases from 1 µm in wild type to 1.8 µm; see Fig. S5). (k and l) Stage 13 embryos expressing Sar1DN in either the endoderm using HkbG4 (k) or mesoderm using TwiG4 (l). In both conditions, midgut migration is delayed; gaps are found between the anterior and posterior midgut rudiments (arrowheads) which have normally fused by stage 13; and the ICPs (asterisks) are found more posteriorly than in wild type (arrows). n = 40 embryos per condition; all embryos show gaps and/or mispositioned ICPs; representative images shown. (m–o) When the CopII pathway is blocked in midgut cells, they form a disorganized-looking monolayer of columnar or wedge-shaped cells, with βPS integrins localized to the apical and basal sides of the cells (n3), although quantification of levels reveals the peaks of βPS to be half that in wild type (o). In contrast, Baz (n1) and E-Cad (n2) are no longer restricted to the apical side of the cells (m–o) and are found in lower levels throughout the lateral membranes (m–o). (p–r) When Sar1DN is expressed in the mesoderm, midgut cells fail to form a monolayered epithelium, remaining rounded and multilayered (p), and Baz (q1) and E-Cad (q2) do not show any polarized localization within the midgut cells. βPS (q3) levels are very reduced, and basal βPS staining appears lost in midgut cells and diffuse throughout the underlying visceral mesoderm (p, q3, and broad basal peak in r). o and r show plots of the average fluorescence intensity (represented as mean gray value) of Baz, E-Cad, and βPS in stage 15 midgut cells measured along the apical (A) to basal (B) axis of a cell (represented in n and q by red line). Each n represents the average of 10 cells measured in one embryo (black dotted lines). n = 6 per condition; the mean for each condition is plotted in red. White dashed lines in d, e, h, i, m, and p indicate the apical side of the midgut, and yellow lines, the basal. Red boxes in m and p depict the area of the midgut epithelium shown in n and q. Confocal images are oriented with the anterior to the left and posterior to the right. Scale bars, 20 µm (a), 50 µm (b, c, k, and l), and 10 µm (d, e, h, i, m, n, p, and q).

Secretion of laminins via the CopII pathway, from both the mesoderm and the midgut cells themselves, is required for their correct migration and MET. (a) Laminins (green) are strongly expressed in the posterior midgut cells in sna,twi mutant embryos. The posterior midgut is demarcated by dashed red lines. (b and c) In stage 13 sar1 zygotic mutants, the ICP cells (asterisks) are found more posteriorly than in wild-type (compare c with wild-type b; the same wild-type was used as in Fig. 2 d for comparison). (d) In the wild-type stage 15 midgut epithelium, two layers of laminin are found on the basal surface, one at the endoderm/mesoderm border (d′, red arrowheads) and the second on the outer surface of the mesoderm (d′, black arrowheads). Additionally, a lower level of LanB2 at the apical surface of midgut cells can be seen (d′, by white dotted line). (e) In stage 15 sar1 mutants, large intracellular punctae of laminins can be found (e, red arrows), indicating defects in laminin secretion (n = 40 embryos at stage 15; all embryos examined had multiple intracellular punctae of LanB2; representative image shown in e). (f and g) Velocity, directional persistence, and coordination values calculated from videos of wild-type (n = 11) and sar1 (n = 6) mutant embryos. n represents the average of a minimum of 15 cells in one embryo (shown as a dot). Data are presented as mean ± SEM. **, P ≤ 0.01 by unpaired two-tailed t test (see Table S1 for raw data). (h) TwiGal4 driving Sar1DN in the mesoderm leads to a loss of LanB2 staining at the endoderm/mesoderm interface (red arrowheads). Low levels of LanB2 staining can be seen throughout the mass of rounded midgut cells (black arrowheads point to the basal side of the visceral mesoderm). (i) HkbGal4 driving Sar1DN in the endoderm leads to a loss of LanB2 staining at the endoderm/mesoderm interface (red arrowheads), while LanB2 can still be seen on the opposite side of the mesoderm (black arrowheads). (j) Plots of the average fluorescence intensity (represented as mean gray value) of LanB2 in stage 15 midgut cells measured along the apical (A) to basal (B) axis of a cell (represented in b by red line). Each n represents the average of 10 cells measured in one embryo (black dotted lines). n = 6 per condition; the mean for each condition is plotted in red. In wild-type cells there is a peak of LanB2 at the endoderm mesoderm border (P1) and on the outer surface of the mesoderm (P2). In TwiG4xSar1DN, LanB2 levels are reduced, P1 appears as a broad diffuse peak, and P2 is either absent or greatly reduced (see Fig. S5 for FWMH). In HkbG4xSar1DN, the endoderm/mesoderm peak, P1, is either absent or greatly reduced, while more diffuse levels of LanB2 can still be seen on the opposite side of the visceral mesoderm (P2 FWMH increases from 1 µm in wild type to 1.8 µm; see Fig. S5). (k and l) Stage 13 embryos expressing Sar1DN in either the endoderm using HkbG4 (k) or mesoderm using TwiG4 (l). In both conditions, midgut migration is delayed; gaps are found between the anterior and posterior midgut rudiments (arrowheads) which have normally fused by stage 13; and the ICPs (asterisks) are found more posteriorly than in wild type (arrows). n = 40 embryos per condition; all embryos show gaps and/or mispositioned ICPs; representative images shown. (m–o) When the CopII pathway is blocked in midgut cells, they form a disorganized-looking monolayer of columnar or wedge-shaped cells, with βPS integrins localized to the apical and basal sides of the cells (n3), although quantification of levels reveals the peaks of βPS to be half that in wild type (o). In contrast, Baz (n1) and E-Cad (n2) are no longer restricted to the apical side of the cells (m–o) and are found in lower levels throughout the lateral membranes (m–o). (p–r) When Sar1DN is expressed in the mesoderm, midgut cells fail to form a monolayered epithelium, remaining rounded and multilayered (p), and Baz (q1) and E-Cad (q2) do not show any polarized localization within the midgut cells. βPS (q3) levels are very reduced, and basal βPS staining appears lost in midgut cells and diffuse throughout the underlying visceral mesoderm (p, q3, and broad basal peak in r). o and r show plots of the average fluorescence intensity (represented as mean gray value) of Baz, E-Cad, and βPS in stage 15 midgut cells measured along the apical (A) to basal (B) axis of a cell (represented in n and q by red line). Each n represents the average of 10 cells measured in one embryo (black dotted lines). n = 6 per condition; the mean for each condition is plotted in red. White dashed lines in d, e, h, i, m, and p indicate the apical side of the midgut, and yellow lines, the basal. Red boxes in m and p depict the area of the midgut epithelium shown in n and q. Confocal images are oriented with the anterior to the left and posterior to the right. Scale bars, 20 µm (a), 50 µm (b, c, k, and l), and 10 µm (d, e, h, i, m, n, p, and q).

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