(A–B’’) Regional breakdown of time-resolved cumulative strain rates, with regions defined at t=0 min based on eccentric pit for the wild-type and a central pit for hkb−/− mutants. For cells ‘near the pit’ in the wild-type (A), tissue constriction dominates (solid gray curves in (A’, A’’)) and is due to isotropic cell constriction (solid green curves in (A’, A’’)), whilst intercalation only plays a minor role in this region (solid orange curves in (A’, A’’)). Cells in this region have completely internalized by about t=20 min. By contrast, in hkb−/− mutant embryos, cells ‘near the (central) pit’ (A), show strongly reduced tissue (dashed gray curves in (A’, A’’)) and cell strain rates (dashed green curves in (A’, A’’)) and mildly reduced intercalation (dashed orange curves in (A’, A’’)). For cells ‘far from the pit’ in the wild-type (B), the tissue elongates toward the pit until t=20 min (solid gray curve in (B’)), with a corresponding contraction circumferentially (solid gray curve in (B’’)), and this is predominantly due to cell intercalation (solid orange curves in (B’, B’’)). Beyond t=20 min, these cells have reached the invagination pit and also constrict isotropically, thereby leading the tissue change (solid gray curves in (B’, B’’) >20 min) to mirror the cell shape change (solid green curves in (B’, B’’) >20 min). By contrast, in hkb−/− mutant embryos, cells ‘far from the (central) pit’ (B) show a slight tissue expansion both radially and circumferentially (dashed gray curves in (B’, B’’)), paired with abnormal circumferential cell elongation (dashed green curves in (B’, B’’)), and some reduced intercalation (dashed orange curves in (B’, B’’)). The corresponding instantaneous strain rate plots can be found in Figure 4—figure supplement 1. Data from nine wild-type movies and five hkb−/− movies were analyzed (see Figure 3—figure supplement 1). (C–E) Quantification of neighbor gains as a measure of T1 and intercalation events. Examples of a circumferential neighbor gain (leading to radial tissue expansion), and a radial neighbor gain (leading to circumferential tissue expansion) are shown in (C). (D) Circumferential neighbor gains dominate over radial neighbor gains in the wild-type (dashed curves), with the rate of neighbor exchanges dropping beyond 20 min. In contrast, in hkb−/− mutant embryos, the amount of circumferential and radial gains is identical (solid curves). (E) Cumulative proportion of productive neighbor gains, defined as the amount of circumferential neighbor gains leading to radial tissue elongation and expressed as a proportion (pp) of cell-cell interfaces tracked at each time point, and split into cells near the pit (eccentric for wild-type and central for hkb−/− mutant) and far from the pit. Predicted productive neighbor gains are strongly reduced and near zero for cells near the pit in hkb−/− mutants compared to control (dashed curves), whereas cells far from the pit in hkb−/− mutant continue to intercalate similar to wild-type (solid curves). (F, G) Myosin II junctional polarity was quantified from segmented and tracked time-lapse movies. Myosin enrichment at junctions can occur in two flavors: Myosin II unipolarity is defined as myosin II enrichment selectively on side of a cell ((F), see schematic inset). Myosin II bi-polarity is defined as myosin II enrichment at two parallel oriented junctions of a single cell, calculated as the magnitude of a vector pointing at the enrichment (G). Data from six wild-type movies and five hkb−/− movies, number of cells is shown in Figure 3—figure supplement 1. Plotted are the rates of change of the uni- and bipolarity amplitudes as a proportion per minute (pp/min) of the mean cell perimeter fluorescence. (F) Circumferential myosin II uni-polar enrichment (i.e., the radial uni-polarity vector, red arrow in schematic, pointing at the myosin enrichment), increases and is high until ~40 min when it drops ((G), green dashed curve). The circumferential uni-polar enrichment is always higher than the radial myosin II uni-polar enrichment (green solid curve in (G)). The myosin II uni-polar enrichment in hkb−/− mutants is overall strongly reduced compared to wild-type (solid curves in (F)). (G) Circumferential myosin II bi-polar enrichment in the wild-type (i.e., the radial bi-polarity vector, red arrow in schematic, pointing at the myosin enrichment) is high until ~40 min when it drops ((G), green dashed curve). Until this point, it is higher than the radial myosin II bi-polar enrichment (green solid curve in (G)). The myosin II bi-polar enrichment in hkb−/− mutants is strongly reduced compared to the wild-type (solid curves in (G)). Statistical significance of p<0.05 (*), p<0.005 (**), p<0.0005 (***) using a mixed-effect model is indicated as shaded boxes at the top and bottom of the panels: comparing circumferential over radial enrichment for either the wild-type or hkb−/− mutants. (H) Analysis of cell shape aspect ratio dynamics in cells far from the pit (eccentric pit for wild-type, cental pit for hkb−/− mutant). In the wild-type, circumferential elongation as part of active circumferential neighbor gains (Sanchez-Corrales et al., 2018) persist until ~t=+20 min, when cells start to become elongated radially (gray curve). In hkb−/− mutants, cells become and remain circumferentially elongated (magenta curve). Data shown for seven wild-type and five hkb−/− mutant movies. (I–L) Analysis of an exemplary cell intercalation event in cells far from the pit in a segmented and tracked time-lapse movie of a hkb−/− mutant placode, stills at the beginning and end of the event shown are in (I), and stills of the whole event in (J). (K) Cells 1 and 2 gain a circumferential contact, mainly via cell elongation. (L) The cell cluster is already circumferentially elongated at t=+14:54 min (black outline) and remains near identically elongated at t=+26:49 min (orange outline). Data for (A–H) pooled from nine wild-type and five hkb−/− mutant movies. In all analyses of the wild-type, t=0 min is defined as the frame just before the first sign of invagination at the future pit was evident. hkb−/− mutants were aligned using as a reference of embryo development the level of invagination of the tracheal pits that are not affected in the hkb−/− mutant as well as other morphological markers such as appearance and depth of segmental grooves in the embryo. Panels (A, A’, B, B’, F, G) are expressed as proportion per minute (pp/min).