βH-spectrin is required for ratcheting apical pulsatile constrictions during tissue invagination (2020)
Figure 3

Figure 3. Medioapical βH-spectrin localization is controlled by the mesoderm-specific transcription factor Twist and Rho signaling activation.

(A) Schematic showing the expression domain of the transcription factor Twist on the ventral side of the Drosophila embryo along the anterior-posterior axis (top: lateral view; bottom: cross section).

(B) Schematic showing the genetic network that induces actomyosin contractility at the apical surface of mesodermal cells. The transcription factors Twist and Snail regulate expression of the G-protein coupled receptor (GPCR) Mist, its ligand Fog and the Gα Cta, as well as T48, which recruits the RhoA activating factor RhoGEF2 to the apical surface. Fog-mediated activation of Mist triggers RhoGEF2 activity through Cta and thus induction of actomyosin contractility.

(C) Schematic of the optogenetic system employed to activate Rho signaling. RhoGEF2-CRY2 is cytosolic in the dark and interacts upon photo-activation with blue light with its binding partner CIBN which is anchored at the plasma membrane.

(D, E) Still frames of a movie showing endogenously tagged mVenus::βH-spectrin in the cross section of a gastrulating wild type (D) and twist mutant (E) embryo imaged using two-photon microscopy. The top frame corresponds to the time point where an initial bending of the apical surface was observed, the middle frame was taken 2 min and the lower frame 3 min afterwards. Scale bars, 30 µm.

(F, G) Confocal images showing apical βH-spectrin (endogenously tagged mVenus::βH-spectrin) in a wild type (F) and twist mutant (G) embryo. Scale bars, 20 μm. Medio-apical βH-spectrin fibers observed in wild type embryos were absent in twist mutant embryos.

(H-Q) Confocal or two-photon microscopy images of the apical surface of an embryo mounted with the dorsal side facing the objective and co-expressing endogenously tagged mVenus::βH-spectrin (green, H, K, M, P), the myosin-II marker Sqh::mCherry (magenta, I, N), and the optogenetic modules as explained in (C). The apical surface is shown before photoactivation (H-L) and 1 min after photoactivation (M-Q). Panels (J, O) show a merge of βH-spectrin and myosin-II and the dashed rectangle indicates regions shown in (L and Q) at higher magnification. Panels (K, L) and (P, Q) are zoom-ins into the region indicated in (J) an (O), respectively. Scale bars, 20 μm (H-J, M-O). 2.5 μm (K, L, P, Q).

(R) Quantification of apical βH-spectrin upon optogenetic stimulation of Rho signaling. The graph shows the mean intensity of βH-spectrin (green) at the apical surface over time as the fold change relative to the initial time point (before photo-activation). The semi-transparent region indicates the standard deviation and dots indicate individual data points (n = 5 embryos).

(S-T) Two-photon microscopy images of apical βH-spectrin (green) 1.5 min after optogenetic stimulation of Rho signaling in embryos that were previously injected with water (S) or the ROCK-inhibitor Y-27634 (T). Dashed boxes indicate photo-activated cells. Y-27634-injected embryos did not undergo apical constriction and did not accumulate βH-spectrin upon photo-activation, while water-injected embryos constricted and showed increased βH-spectrin levels. Scale bars: 10 µm.