Figure 2.

Laminins are required for midgut migration and MET.(a) A dorsal cross-section of a stage 12 wild-type embryo shows that at this stage, hemocytes (red) have not yet migrated to surround the midgut (compare the tip of the migrating hemocytes, arrowheads, with the leading edge of the posterior midgut, arrows). (b) Midgut cells migrate in a highly coordinated fashion, with PMECs on the outside, adjacent to the mesoderm, and the ICPs and AMPs on the inside of a PMEC “sandwich.” (c) After migration, midgut cells take up highly stereotypical positions, ICPs cluster between the anterior and posterior portions of the midgut, the PMECs form a monolayer contacting the visceral muscle, and the AMPs sit on the apical side of the PMECs. (d). In wild-type embryos, the anterior midgut and posterior midgut have met and fused by stage 13, and the ICPs (asterisks) sit in the middle (d, arrow). (e) In LanB1 mutants, the ICPs (asterisks) fail to migrate to the middle and are situated more posteriorly (e, arrow). (f and g) Velocity, directional persistence, and coordination values calculated from videos of wild-type (n = 11) and LanB1 mutant (n = 9) 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 ≤ 0.05; **, P ≤ 0.01 by unpaired two-tailed t test (see Table S1 for raw data). (h) Stage 15 midgut cells in LanB1 mutants are multilayered and rounded. (i) Baz (i1), E-Cad (i2), and βPS (i3) do not show any polarized localization within stage 15 midgut cells. βPS appears diffuse in the visceral mesoderm (i3). (j) Plots of the average fluorescence intensity (represented as mean gray value) of Baz, E-Cad, and βPS in stage 15 LanB1 mutant midgut cells measured along the apical (A) to basal (B) axis of a cell (represented in b by red line). n = 6 embryos per condition (minimum 10 cells per embryo). The sharp peaks of Baz and E-Cad seen in wild-type cells are lost. While apical and basal peaks of βPS are absent, there is a low broad peak of βPS that spans the underlying mesoderm layer, indicating that βPS is down-regulated in midgut cells and delocalized in the mesoderm. White dashed lines in h indicate the apical side of the midgut, and yellow lines, the basal. Red box in h depicts the area of the midgut epithelium shown in i. Confocal images are oriented with the anterior to the left and posterior to the right. Scale bars, 50 µm (a, d, and e) and 10 µm (h and i).