PMC

Intracellular trafficking components are apically enriched in invaginating SGs. (A) A schematic drawing of the anterior region of the Drosophila embryo for stage 11 (ventral and lateral views) and stage 16 (lateral view) with SGs shown in green. Top right, magnified view of a stage 11 SG. The region where SG cells undergo clustered apical constriction during invagination is shown in purple. (B–B’’) En face (top) and lateral (bottom) views of a WT stage 11 SG immunostained for E-Cad, Rab11, and Nuf. Rab11 and Nuf show apical enrichment near the invagination pit (yellow arrowheads). Cyan arrowheads, Rab11 and Nuf signals near the segmental groove. (B’’’–B’’’’’) Heat maps of apical area (B’’’) and intensity of Rab11 (B’’’’) and Nuf (B’’’’’). Cells with smaller apical areas near the invagination pit (dark blue cells in B’’’) show the high intensity of Rab11 and Nuf (red signals in B’’’’ and B’’’’’). (C–C’’’) En face (top) and lateral (bottom) views of a WT stage 11 SG immunostained for E-Cad (C) and Dhc64C (C’). Corresponding heat maps for apical areas (C’’) and intensity of Dhc64C signals (C’’’) are shown. (D–F’’’) WT stage 11 SGs immunostained for E-Cad and Rab11 at different timepoints of invagination. (G) The total intensity of Rab11 in the whole SG placode before and during invagination. Before invagination, n = 4 SGs; during invagination, n = 5 SGs. (H) Negative correlation between Rab11 intensities and apical areas of SG cells before (top) and during invagination (bottom). R, Pearson correlation coefficient. P < 0.0001 for all samples. Before invagination, n = 4 SGs, 506 cells; during invagination, n = 4 SGs, 561 cells. Asterisks: invagination pit. White lines: SG boundary. SG boundaries are marked based on CrebA (SG nuclear marker) signals in the basal region of SG cells.

Wild-type expression of hedgehog (hh) and wingless (wg) in Oncopeltus, before and after blastoderm invagination. (A) Expression of hh at 32 h after egg laying (hAEL), late blastoderm stage, is as a ring in the anterior third of the embryo. (B) At around 36 hAEL, the hh anterior expression ring splits into two stripes corresponding to the presumed ocular and antennal segments. Six additional stripes of expression appear, corresponding to the presumed gnathal and thoracic segments. (C) Embryo at mid-invagination, ∼42 hAEL. The two anterior stripes are fully separated. Thoracic segments have already invaginated and expression in them can be seen through the yolk. (D,E) Germband embryos, ∼48–52 hAEL. In the later germband embryo, ∼52 hAEL (E), two dots of hh expression appear in the presumed intercalary segment, which are not seen in the earlier embryo (D). (F) Expression of wg at ∼32 hAEL is located to an anterior stain and a posterior stain marking the future invagination site. (G) At ∼36 hAEL seven stripes of expression appear simultaneously, corresponding to the presumed antennal, gnathal, and thoracic segments. (H) Embryo at mid-invagination, ∼42 hAEL. This embryo is slightly older than the one in C as evidenced by the extent of invagination. (I,J) Germband embryos, ∼48–52 hAEL. In the later germband embryo, ∼52 hAEL (J), two dots of wg expression appear in the presumed intercalary segment, which cannot be seen in the earlier embryo (I). (K,M) The process of invagination from ∼36–42 hAEL. Nuclei stained by SYTOX in cyan and wild-type wg expression in red. When invagination begins (K), the blastoderm is still mostly uniform. At mid-invagination (L) most blastoderm cells migrate dorsally, with the anterior cells, corresponding to the future head, condensing to give a head lobe. At late invagination (M) the head lobe is distinct. At this stage, the anterior-posterior axis of the egg is opposite to that of the invaginating embryo. (N,O) Illustration of the invagination process. “A” marks the embryonic anterior and “P” the posterior. The portions of the embryo that have invaginated to give the germband are shown in darker green. Panel (N) corresponds to the embryo in (L) and panel (O) corresponds to the embryo in (M). at, antennal segment; ic, intercalary segment; lb, labial segment; oc, ocular segment; md, mandibular segment; mx, maxillary segment; T1–T3, first to third thoracic segments. Axis labels: A, anterior; P, posterior; D, dorsal; V, ventral. All scale bars are 200 μm.

An apico-basal force drives mesoderm invagination. Schemes recapitulate the orientation of the embryos and the laser cuts performed in each experiment. a Longitudinal views of sqhKI[eGFP] control embryos (n = 17), showing the linear shape of the invagination front at early stage. b (top) Longitudinal views of sqhKI[eGFP] embryo ablated in the mid-plane region (red rectangle in the scheme and a dashed magenta line in the figure, n = 12), showing a slight delay in the central region compared to the anterior one, as shown by the outline of the invagination front in magenta at early stage of invagination. (bottom) Scheme of the invagination fronts in the control (white line, as in a) and ablated embryo (magenta line). c Anterior (left) and central (right) transversal views of a mid-plane ablated embryo, at two different times of the invagination. Note that invagination proceeds normally as shown by the V-shaped invagination outlined by a white dotted line (bottom), although a delay in the central region can be observed at early stage (top, compare anterior to central region). d Ventral and transversal views extracted from a movie of a control sqhKI[eGFP] embryos, showing the different step of mesoderm invagination. e The apical surface was cut on the anterior and the posterior sides (blue arrowheads, see left schemes) to isolate the central domain of the mesoderm. Note that the invagination is taking place normally, in term of invagination shape, depth and timing (n = 10). f After isolation of the central domain (apical cuts as described in  e), ablation was performed in the mid-plane of the central region of the mesoderm (red rectangle on the scheme, magenta dashed line in the figure). Note that the invagination is totally prevented when apico-basal forces are impaired in the vast majority of the cases (n = 11/12). e, f Note that apical constriction occurs in both cases, as shown by the curved shape adopted by the ventral surface (white arrowheads). Apico-basal cables are not visible due to low resolution. Scale bar: 10 µm. An anterior, P posterior, V ventral, D dorsal, Ap apical, B basal. See also Supplementary Figs. , and Supplementary Movie 

Four ectodermal cells near the ventral furrow were indicated in a Late Group embryo. No obvious AP movement was observed, which is consistent with the notion that ventral furrow invagination in Late Group embryos is not attributed to germband extension. Shown is a representative embryo of six Late Group embryos analyzed. Time zero corresponds to the onset of gastrulation. Scale bars: 20 μm.