Figure 4.
Figure 4. Actin cross-linkers Cheerio and Fimbrin display distinct regulatory functions during the hexagonal and the ring phase. (A–H) Confocal sections at the level of the basal actomyosin network during sequential stages of cellularization in embryos coexpressing endogenously tagged Cheerio::YFP (A and C) or Fimbrin::YFP (E and G) and the myosin-II probe Sqh::mCh (magenta; B, D, F, and H). Top view during the hexagonal phase at an ingression depth of 5 µm (A, B, E, and F). Top view during the ring phase at an ingression depth of 12 µm (C, D, G, and H). Both Cheerio and Fimbrin colocalized with myosin-II at the cell base during all stages of cellularization. Scale bars, 10 µm. (I–P) Confocal images illustrating the actomyosin network in cellularizing embryos expressing the myosin-II marker Sqh::mCh in control embryos (I and J), in bnk−/− embryos (K and L), and in embryos expressing the nanobody KD module targeting Fimbrin (M and N) or Cheerio (O and P). The upper panels show sagittal cross sections (scale bars, 20 µm) and the lower panels the top view of basal myosin-II signal. Scale bars, 10 µm. Images represent two sequential stages of cellularization: furrow ingression is at 5 µm from the apical surface (I, K, M, and O), and furrow ingression is at 10 µm from the apical surface (J, L, N, and P). In control embryos, the actomyosin network displayed either a hexagonal (I) or round (J) conformation. In bnk−/− or Cheerio KD embryos, the network was disordered and did not acquire the hexagonal configuration (K and O). In contrast, upon Fimbrin KD, the hexagonal phase was prolonged, and the transition into contractile rings was delayed (M). (Q and R) Quantification of geometric features of actomyosin network with respect to the ingression depth of the cellularization furrow. The average angularity (Q) and area (R) of cell bases in either a control (black), bnk−/− (red), Cheerio KD (green), or Fimbrin KD (blue) embryo were plotted over the ingression depth. (Q) In the control, actomyosin fibers assembled in hexagonal units of high angularity. At an invagination depth of the furrow of ∼7 µm, the hexagonal units rounded up, displaying angularity values closer to 1. In bnk−/− and Cheerio KD embryos, actomyosin fibers formed round units (angularity close to 1) from the beginning of the cellularization process. In contrast, the actomyosin network in a Fimbrin KD maintained an angular shape until an ingression depth of ∼15 µm. (R) In bnk−/− and in Cheerio KD embryos, individual cell bases constricted prematurely at a lower ingression depth compared with control and displayed a smaller area. In the Fimbrin KD condition, network constriction was delayed compared with controls. (Q and R) Solid lines indicate average values and semitransparent areas the SEM. For each condition, individual cell openings were segmented and analyzed (basal openings per data point: 27 ≤ ncontrol ≤ 87, 26 ≤ nbnk−/− ≤ 162, 15 ≤ nCheerioKD ≤ 42, 21 ≤ nFimbrinKD ≤ 50). (S) Bar diagram showing the penetrance of Cheerio and Fimbrin KD phenotypes. Cheerio KD (n = 30), Fimbrin KD (n = 16), and control embryos (NControl Cheerio = 35, NControl Fimbrin = 19) were scored for the prevalence of the different phenotypes in blind tests. White color indicates the percentage of embryos with no or very mild phenotypes; red indicates mild or strong phenotypes.

Actin cross-linkers Cheerio and Fimbrin display distinct regulatory functions during the hexagonal and the ring phase. (A–H) Confocal sections at the level of the basal actomyosin network during sequential stages of cellularization in embryos coexpressing endogenously tagged Cheerio::YFP (A and C) or Fimbrin::YFP (E and G) and the myosin-II probe Sqh::mCh (magenta; B, D, F, and H). Top view during the hexagonal phase at an ingression depth of 5 µm (A, B, E, and F). Top view during the ring phase at an ingression depth of 12 µm (C, D, G, and H). Both Cheerio and Fimbrin colocalized with myosin-II at the cell base during all stages of cellularization. Scale bars, 10 µm. (I–P) Confocal images illustrating the actomyosin network in cellularizing embryos expressing the myosin-II marker Sqh::mCh in control embryos (I and J), in bnk−/− embryos (K and L), and in embryos expressing the nanobody KD module targeting Fimbrin (M and N) or Cheerio (O and P). The upper panels show sagittal cross sections (scale bars, 20 µm) and the lower panels the top view of basal myosin-II signal. Scale bars, 10 µm. Images represent two sequential stages of cellularization: furrow ingression is at 5 µm from the apical surface (I, K, M, and O), and furrow ingression is at 10 µm from the apical surface (J, L, N, and P). In control embryos, the actomyosin network displayed either a hexagonal (I) or round (J) conformation. In bnk−/− or Cheerio KD embryos, the network was disordered and did not acquire the hexagonal configuration (K and O). In contrast, upon Fimbrin KD, the hexagonal phase was prolonged, and the transition into contractile rings was delayed (M). (Q and R) Quantification of geometric features of actomyosin network with respect to the ingression depth of the cellularization furrow. The average angularity (Q) and area (R) of cell bases in either a control (black), bnk−/− (red), Cheerio KD (green), or Fimbrin KD (blue) embryo were plotted over the ingression depth. (Q) In the control, actomyosin fibers assembled in hexagonal units of high angularity. At an invagination depth of the furrow of ∼7 µm, the hexagonal units rounded up, displaying angularity values closer to 1. In bnk−/− and Cheerio KD embryos, actomyosin fibers formed round units (angularity close to 1) from the beginning of the cellularization process. In contrast, the actomyosin network in a Fimbrin KD maintained an angular shape until an ingression depth of ∼15 µm. (R) In bnk−/− and in Cheerio KD embryos, individual cell bases constricted prematurely at a lower ingression depth compared with control and displayed a smaller area. In the Fimbrin KD condition, network constriction was delayed compared with controls. (Q and R) Solid lines indicate average values and semitransparent areas the SEM. For each condition, individual cell openings were segmented and analyzed (basal openings per data point: 27 ≤ ncontrol ≤ 87, 26 ≤ nbnk−/− ≤ 162, 15 ≤ nCheerioKD ≤ 42, 21 ≤ nFimbrinKD ≤ 50). (S) Bar diagram showing the penetrance of Cheerio and Fimbrin KD phenotypes. Cheerio KD (n = 30), Fimbrin KD (n = 16), and control embryos (NControl Cheerio = 35, NControl Fimbrin = 19) were scored for the prevalence of the different phenotypes in blind tests. White color indicates the percentage of embryos with no or very mild phenotypes; red indicates mild or strong phenotypes.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal